RU2209634C1 - Kit "estrogen-receptor" for detecting estrogen-receptors in bioptic histological preparations - Google Patents

Abstract

FIELD: analytical methods in medicine. SUBSTANCE: invention provides two detection variants. Kit contains monoclonal antibodies against beta-chain of estrogen specially prepared for immunochemical staining (IgG, Sigma AER311) and standard monoclonal anti-IgG antibodies conjugated with bioptin (Sigma) also intended for immunochemical coloration. Enzymes catalyzing formation of stained product registered by light microscopy technique are peroxidase and alkali phosphatase. In particular, alkali phosphatasestreptavinin complex is utilized. EFFECT: enhanced diagnostic efficiency and enabled staining of different tissues with different properties. 2 dwg, 1 tbl

Description

 The invention relates to the field of medicine.

 Estrogens are a family of steroid hormones that regulate the vital activity of the human body by acting on a number of target organs, primarily organs of the reproductive system. The action of estrogens on the human body is carried out through the stage of binding of the hormone to a molecule of a specific protein - the estrogen receptor (ER), which after such binding to the hormone is able to activate the genetic apparatus of the cell and realize the biological activity of the hormone.

 The need to determine ER in human cells is primarily due to the fact that a number of malignant tumors, including a very common breast cancer, can be hormone-sensitive, i.e., the growth of hormone-sensitive forms of tumors is accelerated by the action of estrogen ( see, for example, Saunders RT, Millar MR, Williams K, Macpherson S, Bayne C, O'Sullivan C, Anderson TJ, Groome NP, Miller W. Expression of oestrogen receptor beta (ERbetal) protein in human breast cancer biopsies. Br J Cancer 2002 Jan 21; 86 (2): 250-6).

 The effect of many antitumor drugs is based on this sensitivity, however, before such drugs are used, it is necessary to make sure that the tumor has hormone sensitivity. Since in the overwhelming majority of cases the cause of numbness is precisely the absence of ER, the determination of this receptor using a test kit gives an answer to the question of the possibility of using antitumor drugs that act through ER, i.e. allows you to choose the right treatment.

The principle of operation of the kit is based on the following:
- the interaction of ER molecules (on microscopic sections) with specific antibodies;
- accession to specific antibodies modified with biotin, a complex of streptavidin and alkaline phosphatase;
- obtaining a colored alkaline phosphatase product and its registration using a light microscope.

 The principle of the specific interaction of antibodies with antigens is a fundamental principle of the physical chemistry of proteins and immunochemistry. In general, it is described in all manuals on immunology and immunochemistry, for example, "Immunology", A. Royt, J. Brostoff, D. Mail, Mir, 2000.

 As applied to receptors for steroid hormones and, in particular, to estrogens, it is described: Dabbs DJ, Landreneau RJ, Liu Y, Raab SS, Maley RH, Tung MY, Silverman JF. Detection of estrogen receptor by immunohistochemistry in pulmonary adenocarcinoma. Ann Thorac Surg. 2002 Feb; 73 (2): 403-5.

 The principle of using a biotin modification with subsequent attachment of the enzyme and obtaining a colored product is also generally accepted; as applied to ER, it is described: McCluggage WG, Sumathi VP, McBride HA, Patterson A. A panel of immunohistochemical stains, including carcinoembryonic antigen, vimentin, and estrogen receptor, AIDS the distinction between primary endometrial and endocervical adenocarcinomas. Int J Gynecol Pathol. 2002 Jan; 21 (l): 11-5.

 The main non-immunochemical method for determining the expression of ER is based on the use of reverse transcription followed by polymerase chain reaction (PCR). This method has significant drawbacks. First, the PCR method makes it possible to analyze the expression of the ER gene at the level of mRNA formation, but the active ER protein may not form in the cell in the presence of normal mRNA. Secondly, the reverse transcription PCR method requires RNA isolation, which is very laborious, technically difficult and generally not applicable for large-scale diagnostic assays. Both of these disadvantages are absent in the method for determining ER proposed in this patent.

 Currently, Russia does not produce kits for the immunochemical determination of ER, the closest of the kits produced outside Russia is the DAKO ER / PR System test kit from Dako, Denmark (kat. # 1900 from the 2002 catalog).

 The objective of the present invention is to provide a domestic kit for the immunochemical determination of estrogen receptor, intended for diagnostic purposes (see table 1).

 To solve this problem, a group of inventions is proposed, united by a common inventive concept and representing options.

 1st option.

A kit for detecting estrogen receptors (ERs) in biopsy histological specimens contains
- solution restoring the structure of the antigen, 10x-concentrate;
- primary antibodies, which are a solution of murine monoclonal antibodies to ER in 0.05 M Tris-Hcl buffer, pH 7.6, containing bovine serum albumin and 15 mM sodium azide;
- secondary antibodies, which are a solution of murine monoclonal biotin-labeled antibodies to mouse immunoglobulin in 0.05M Tris-Hcl buffer, pH 7.6, containing bovine serum albumin and 15 mM sodium azide;
- streptavidin peroxidase, which is streptavidin conjugated to horseradish peroxidase in PBS containing bovine serum albumin and 15 mM sodium azide;
- 3% hydrogen peroxide solution;
- imidazole-Hcl buffer, pH 7.5, containing hydrogen peroxide and 15 mM sodium azide;
- DAB-chromogenic substrate: 3,3'-diaminobenzidine in a chromogen solution;
- a washing solution containing a mixture of salts per 1 liter of Tris-Hcl 50 mM, pH 8.0; NaCl 150 mM.

 2nd option.

A kit for detecting estrogen receptors (ERs) in biopsy histological specimens contains
- solution restoring the structure of the antigen, 10x-concentrate;
- primary antibodies, which are a solution of murine monoclonal antibodies to ER in 0.05 M Tris-Hcl buffer, pH 7.6, containing bovine serum albumin and 15 mM sodium azide;
- secondary antibodies, which are a solution of murine monoclonal biotin-labeled antibodies to mouse immunoglobulin in 0.05 M Tris-Hcl buffer, pH 7.6, containing bovine serum albumin and 15 mM sodium azide;
- buffer detector, Sigma A4955;
- Streptavidin, a solution of 1 mg / ml, a solution in the buffer detector;
- Biotinylated alkaline phosphatase, 1 mg / ml, solution in
- buffer detector;
- NBT-BCIP - reagent, Sigma, In 1911;
- reaction buffer, including Tris-HCl 50 mM, pH 9.5; NaCl 100 mM, MgCl ₂ 1 mM, NaN ₃ 0.1%, levamisole 0.1 mM;
- a washing solution containing a mixture of salts per 1 liter of Tris-Hcl 50 mM, pH 8.0; NaCl 150 mM.

In the proposed set of "ESTROGEN-RECEPTOR":
- used new monoclonal antibodies to the beta chain of ER, specially obtained for immunochemical staining (IgG, Sigma AER311) and standard monoclonal antibodies against IgG conjugated with biotin, manufactured by Sigma, also intended for immunochemical staining;
- used not only peroxidase, but also alkaline phosphatase as an enzyme giving a stained product detected by light microscopy, which gives more opportunities for optimizing staining of preparations from various tissues with different properties;
- the alkaline phosphatase-streptavidin complex was used, and streptavidin and alkaline phosphatase are included separately in the kit, this makes it possible to change their ratio to optimize the determination of ER;
- unlike the PathoGene kit, the ESTROGEN RECEPTOR is intended for diagnostic purposes.

 COMPOSITION OF THE SET.

 1) Solution restoring the structure of the antigen: 10x-concentrate. 100ml

 2) Primary antibodies: 7 ml of a solution of murine monoclonal antibodies to the estrogen receptor (ER) in 0.05 M Tris-Hcl buffer, pH 7.6, containing bovine serum albumin and 15 mM sodium azide.

 3) Secondary antibodies: 7 ml of a solution of murine monoclonal biotin-labeled antibodies to mouse immunoglobulin in 0.05 M Tris-Hcl buffer, pH 7.6, containing bovine serum albumin and 15 mM sodium azide.

 4) Streptavidin-Peroxidase: streptavidin conjugated to horseradish peroxidase in PBS containing bovine serum albumin and 15 mM sodium azide.

 5) Hydrogen peroxide: 3% hydrogen peroxide solution. 15 ml

 6) Imidazole-HCl buffer, pH 7.5, containing hydrogen peroxide and 15 mM sodium azide.

 7) DAB-chromogenic substrate: 3,3'-diaminobenzidine in a chromogen solution.

 8) Buffer detector, 10 ml. Sigma, A4955.

 9) Streptavidin, a solution of 1 mg / ml, a solution in the detector buffer (8), 2 ml.

 10) Biotinylated alkaline phosphatase, 1 mg / ml, solution in the detector buffer (8), 1 ml.

 11) NBT-BCIP - reagent, 200 μl. Sigma, In 1911.

12) Reaction buffer, 10 ml:
Tris-HCl 50 mM, pH 9.5; NaCl 100 mM, MgCl ₂ 1 mM, NaN ₃ 0.1%, levamisole 0.1 mM.

13) A mixture of salts for washing solution - 1 l:
Tris-Hcl 50 mM, pH 8.0; NaCl 150 mM.

 SET APPLICATION TECHNIQUE.

 Preparation and pre-treatment of histological sections.

Prepare paraffin sections of the histological material with a thickness of 4-6 microns. It is necessary to use highly adhesive glass, pre-treated with a solution of polylysine or silane. Slices are dried in an upright position for 18 hours at a temperature of 60-80 ^o C. If necessary, the finished sections are stored at 4 ^o C.

Next, the sections are kept at a temperature of 60-80 ^o C for 5-15 minutes and dewaxing is carried out:
- Xylene 10 min
- Xylene 2 min
- 100% alcohol 1 min
- 100% alcohol 1 min
- 96% alcohol 1 min
- 70% alcohol 1 min
- 50% alcohol 1 min
- Distilled water 1 min.

Then, at room temperature, the sections are dehydrated according to the following scheme:
- H ₂ O distilled - 1 min
- 50% alcohol - 1 min
- 70% alcohol - 1 min
- 96% alcohol - 1 min
and dried sections at a temperature of 37 ^o C for 5-10 minutes If necessary, prepared sections can be stored at 4 ^o C for up to 1 week.

 Staining slices.

 The material is heat treated according to standard histochemical methods using a pressure cooker or an autoclave. For processing using a solution that restores the structure of the antigen. A working solution that restores the antigen structure is prepared by diluting 10 times the 10x concentrate included in the kit (1). Apply 100 μl of hydrogen peroxide solution (5) to the samples and incubate for 5 minutes.

 Wash samples with wash solution (13). Apply a solution of primary antibodies (2) and incubate for 10 minutes. Wash samples with wash solution (13). Apply a solution of secondary antibodies (3) and incubate for 10 minutes. Wash samples with wash solution (13).

 Next, you should use one of the two methods, depending on the selected enzyme. Option 1. Staining sections using peroxidase.

 1. Apply Streptavidin-Peroxidase solution: (4) and incubate for 10 minutes.

 2. Wash the samples with wash solution (13).

 3. A substrate solution is prepared, for which 20 μl of solution 7 is added to 1 ml of buffer 6 and mixed thoroughly. Apply a substrate solution to the samples and incubate for 5 minutes.

 4. Wash the samples with wash solution (13) and distilled water.

 Option 2. Staining sections using alkaline phosphatase.

1. Apply a reagent detector of 100 μl to the sections and incubate in a humid chamber at 37 ^° C for 30 minutes.

To prepare the detector reagent, a streptavidin solution (9), 1: 5 by volume and a solution of biotinated alkaline phosphatase (10), 1:10 by volume, are added to the buffer detector (8), carefully mixed and kept at 37 ^° C for 30 minutes.

 After this, the finished reagent detector can be applied to the slices.

 2. The sections are washed 3 times with washing solution (13) for 1 min.

3. Apply 100 μl NBT / BCIP reagent solution to the sections per section and incubate in a humid chamber in the dark! at 37 ^o C for 30 minutes

For the preparation of NBT / BCIP reagent
A 2% NBT / BCIP reagent is prepared in the reaction buffer (12); the prepared solution must be protected from direct sunlight.

 4. The sections are washed 3 times with washing solution (13) for 1 minute.

 5. Wash sections with distilled water for 1 min.

Sections stained with peroxidase or alkaline phosphatase dehydrate
- 96% alcohol 1 min
- 100% alcohol 1 min
- 100% alcohol anhydrous 1 min
- xylene 1 min
and enclose in a Canadian balm (or other medium) under a coverslip. The drug is ready for examination under a light microscope.

Claims (2)

 1. A kit for the detection of estrogen receptors (ER) in biopsy histological specimens, characterized in that it contains a solution that restores the structure of the antigen, 10x-concentrate; primary antibodies, which are a solution of murine monoclonal antibodies to ER in 0.05 M Tris-HCl buffer, pH 7.6, containing bovine serum albumin and 15 mM sodium azide; secondary antibodies, which are a solution of murine monoclonal biotin-labeled antibodies to mouse immunoglobulin in 0.05 M Tris-HCl buffer, pH 7.6, containing bovine serum albumin and 15 mM sodium azide; streptavidin peroxidase, which is streptavidin conjugated to horseradish peroxidase in PBS containing bovine serum albumin and 15 mM sodium azide; 3% hydrogen peroxide solution; imndazole-HC1 buffer, pH 7.5, containing hydrogen peroxide and 15 mM sodium azide; DAB-chromogenic substrate, 3,3'-diaminobenzidine in a chromogen solution; a washing solution containing a mixture of salts per 1 liter of Tris-HC1 50 mM, pH 8.0; NaCl 150 mM. 2. A kit for the detection of estrogen receptors (ER) in biopsy histological specimens, characterized in that it contains a solution that restores the structure of the antigen, 10x-concentrate; primary antibodies, which are a solution of murine monoclonal antibodies to ER in 0.05 M Tris-HCl buffer, pH 7.6, containing bovine serum albumin and 15 mM sodium azide; secondary antibodies, which are a solution of murine monoclonal biotin-labeled antibodies to mouse immunoglobulin in 0.05 M Tris-HCl buffer, pH 7.6, containing bovine serum albumin and 15 mM sodium azide; buffer detector, Sigma A4955; Streptavidinum, solution of 1 mg / ml, solution in the buffer detector; biotinylated alkaline phosphatase, 1 mg / ml, solution in the detector buffer; NBT-BCIP - reagent, Sigma, B1911; reaction buffer, including Tris-HC1 50 mM, pH 9.5; NaCl 100 mM, MgCl ₂ l mM, NaN ₃ 0.1%, levamisole 0.1 mM; a washing solution containing a mixture of salts per 1 liter of Tris-HC1 50 mM, pH 8.0; NaCl 150 mM.

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