CN212989384U

CN212989384U - Immunohistochemical detection kit for P16, MCM2 and Ki67

Info

Publication number: CN212989384U
Application number: CN202021116160.XU
Authority: CN
Inventors: 姚远颋; 许舟; 顾重建; 李克强
Original assignee: Chongqing Wokang Biological Technology Co ltd
Current assignee: Chongqing Wokang Biological Technology Co ltd
Priority date: 2020-06-16
Filing date: 2020-06-16
Publication date: 2021-04-16
Anticipated expiration: 2030-06-16

Abstract

The utility model discloses a P16 and MCM2, Ki67 immunohistochemical detection kit, including box body and first aseptic device hole, the laminating of box body inner wall has the liner, and liner upper surface edge is provided with first container hole, first container hole one side distributes and has second container hole, and second container hole one side distributes and have third container hole, third container hole one side distributes and has fourth container hole, first container hole right side distributes and has fifth container hole, and fifth container hole one side distributes and has sixth container hole. This P16 and MCM2, Ki67 immunohistochemical detect reagent box is provided with first container hole, second container hole, reagent bottle has all been placed to third container hole and fourth container hole inside, and the splendid attire has a P16 anti concentrate in the reagent bottle respectively, Ki-67 anti concentrate, MCM2 anti concentrate and antibody diluent, P16 and MCM2, Ki-67 immunohistochemical three staining of dying detect high-efficiently, and is stable, can be fine satisfy the science, the needs of work.

Description

Immunohistochemical detection kit for P16, MCM2 and Ki67

Technical Field

The utility model relates to an immune tissue detection kit technical field specifically is P16 and MCM2, Ki67 immunohistochemical detection kit.

Background

The p16 gene is also known as a multiple tumor suppressor gene (MTS), and the expressed cancer suppressor protein p16 is a cell cycle progression negative regulatory protein. When the human papilloma virus is integrated and infects cervical epithelial cells, an oncogene E7 is introduced and activated, so that the expression of an oncogenic protein E7 is promoted, pRB is inhibited from being combined with E2F protein, and the proliferation of the cervical epithelial cells is promoted; when the cell is over-proliferated, a negative feedback regulation mechanism of the cell proliferation is activated, namely, the cancer suppressor gene p16 is activated to promote the expression of the cancer suppressor protein p16 and promote the combination of pRB and E2F protein, so that the over-proliferation of the cervical epithelial cells is inhibited.

MCM2 and Ki-67 genes are related to cell proliferation, the expression of the genes is different according to different cell cycle phases, the genes begin to be expressed in the G1 phase of the cell cycle, the expression is increased in the S phase and the G2 phase, the expression reaches the peak in the M phase, the genes are rapidly degraded or lose antigenic determinants in the later stage of cell mitosis, and the genes are not expressed in the G0 phase. The half-life period is short, so that the cell growth fraction is not easy to induce by growth factors, and the cell growth fraction can be used as an index for evaluating the cell growth fraction.

The existing detection kit detects by single staining of immunohistochemistry, namely, a single-pass immunohistochemistry method only detects the P16 gene or only detects MCM2 and Ki-67 genes.

In the use of the immune tissue detection kit in the market, in cervical cancer screening, only the P16 gene or only the MCM2 and Ki-67 gene is detected by a single immunohistochemical method, the detection is time-consuming and labor-consuming, two samples need to be processed simultaneously, and then the detection is carried out to obtain a result, so that the detection is complex, the staining solution is easy to become old, the staining effect is unstable, the accuracy of pathological interpretation of experimenters is influenced, the observation is inconvenient, the detection accuracy is low, the two-time single staining is time-consuming and labor-consuming, but if the three staining is combined, a large number of reagents are required to be assembled in the same kit. The structure of the kit needs to be improved, and the same three-staining kit has the advantages that the buffer solution is divided into two tubes for subpackage due to larger filling amount, so that the possibility of pollution is caused, but the buffer solution with larger filling amount in one tube can increase the volume of the kit, and therefore, the P16 and MCM2 and Ki67 immunohistochemical detection kits are provided.

SUMMERY OF THE UTILITY MODEL

An object of the utility model is to provide P16 and MCM2, Ki67 immunohistochemical detect reagent box, in order to solve the cervical carcinoma screening that proposes in the above-mentioned background art, the method of single through immunohistochemical only detects P16 gene or only detects MCM2, the method of Ki-67 gene, it wastes time and energy to detect, need handle two samples simultaneously, then detect and obtain the result, comparatively complicated, the dyeing liquid is easy old and old, and dyeing effect is unstable, influence the accuracy of experimenter's pathology slide, be not convenient for observe, the rate of accuracy of detection is low, it is hard to dye twice singly, but if the triplex dyeing combines, just need a large amount of reagent to assemble in same kit. The structure of the kit needs to be improved, and in the same three-dye kit, the buffer solution is divided into two tubes for subpackage due to large loading amount, which causes the possibility of pollution, but the buffer solution with large loading amount in one tube can increase the volume of the kit.

In order to achieve the above object, the utility model provides a following technical scheme: the immunohistochemical detection kit for P16, MCM2 and Ki67 comprises a box body and a first sterile device hole, wherein a liner is attached to the inner wall of the box body, and the edge of the upper surface of the liner is provided with a first container hole, one side of the first container hole is distributed with a second container hole, a third container hole is distributed on one side of the second container hole, a fourth container hole is distributed on one side of the third container hole, a fifth container hole is distributed on the right side of the first container hole, and a sixth container hole is distributed on one side of the fifth container hole, a seventh container hole is distributed on one side of the sixth container hole, and an eighth container hole is distributed on one side of the seventh container hole, the first aseptic device hole is distributed on the right side of the eighth container hole, and a second sterile device hole is distributed on the right side of the first sterile device hole, a first hinge is arranged at one end of the first sterile device hole, and a second hinge is arranged at one end of the second sterile device hole.

Preferably, the inner wall of the box body is attached to the outer wall of the gasket, and the vertical central axis of the box body coincides with the vertical central axis of the gasket.

Preferably, the horizontal central axes of the first container hole, the second container hole, the third container hole and the fourth container hole coincide, and the first container hole and the fourth container hole are symmetrically distributed about the horizontal central axis of the gasket.

Preferably, the horizontal central axis of the fifth container hole, the horizontal central axis of the sixth container hole, the horizontal central axis of the seventh container hole and the horizontal central axis of the eighth container hole coincide, and the fifth container hole and the eighth container hole are symmetrically distributed about the horizontal central axis of the gasket.

Preferably, the first sterile device hole is parallel to the horizontal central axis of the second sterile device hole, and the length and width of the first sterile device hole are equal to the length and width of the second sterile device hole.

Preferably, the first hinge is movably connected with the first sterile device hole, and the first hinge is overlapped with a vertical central axis of the first sterile device hole.

Compared with the prior art, the beneficial effects of the utility model are that: this P16 and MCM2, Ki67 immunohistochemical detect reagent box is provided with first container hole, first container hole is the symmetric distribution with fourth container hole about the horizontal axis of liner, first container hole, the second container hole, reagent bottle has all been placed to third container hole and fourth container hole inside, and contain respectively in the reagent bottle and be a primary anti concentrate of P16, a Ki-67 primary anti concentrate, an anti concentrate of MCM2 and antibody diluent, P16 and MCM2, the immunohistochemical three staining of Ki-67 detects high-efficiently, and is stable, can be fine satisfy the needs of science, work.

The fifth container hole and the eighth container hole are symmetrically distributed about a horizontal central axis of the liner, reagent bottles are respectively placed inside the fifth container hole, the sixth container hole, the seventh container hole and the eighth container hole, goat anti-mouse secondary antibody working solution, goat anti-rabbit secondary antibody working solution, DAB staining substrates and DAB staining substrates are respectively contained in the reagent bottles, a user can conveniently perform immunohistochemical staining tests, and immunohistochemical tertiary staining of P16, MCM2 and Ki-67 is performed. The kit is superior to similar immunohistochemical single staining kit for detecting P16 gene or MCM2 and Ki-67 gene only. The results of the immunohistochemical triple staining methods of P16, MCM2 and Ki-67 are displayed on the same sample slide, namely the results are displayed on the same sample slide, so that the accuracy of pathological slide reading of experimenters is improved, the observation is convenient, the detection accuracy is improved, the possibility of false positive and false negative is reduced, and the possibility of misjudgment is reduced.

First loose-leaf and the coincidence of the vertical axis in first aseptic device hole, through this structure, the user need mention first loose-leaf, second loose-leaf then take out the reagent bottle and use, and the user operation is more convenient.

Drawings

FIG. 1 is a schematic view of the overall structure of the present invention;

FIG. 2 is a schematic view of a first aseptic device well structure of the present invention;

fig. 3 is a schematic view of a first hinge structure of the present invention.

In the figure: 1. a box body; 2. a liner; 3. a first container aperture; 4. a second container aperture; 5. a third container aperture; 6. a fourth container aperture; 7. a fifth receptacle well; 8. a sixth container aperture; 9. a seventh receptacle well; 10. An eighth container aperture; 11. a first sterile device aperture; 12. a second sterile device aperture; 13. a first leaflet; 14. and a second leaflet.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by a person skilled in the art without creative work belong to the protection scope of the present invention.

Referring to fig. 1-3, the present invention provides a technical solution: the kit comprises a box body 1, a gasket 2, a first container hole 3, a second container hole 4, a third container hole 5, a fourth container hole 6, a fifth container hole 7, a sixth container hole 8, a seventh container hole 9, an eighth container hole 10, a first sterile device hole 11, a second sterile device hole 12, a first hinge 13 and a second hinge 14, wherein the gasket 2 is attached to the inner wall of the box body 1, the inner wall of the box body 1 is attached to the outer wall of the gasket 2, the vertical central axis of the box body 1 is overlapped with that of the gasket 2, and the structure is arranged so that the kit is compact in structure, convenient to carry, good in repeatability, time-saving and labor-saving, and the gasket 2 can buffer impact on a reagent bottle in the box body 1 when the device shakes, so that the safety of the reagent bottle is ensured, and the requirements of a user are met;

the edge of the upper surface of the gasket 2 is provided with a first container hole 3, one side of the first container hole 3 is distributed with a second container hole 4, one side of the second container hole 4 is distributed with a third container hole 5, one side of the third container hole 5 is distributed with a fourth container hole 6, the horizontal central axes of the first container hole 3, the second container hole 4, the third container hole 5 and the fourth container hole 6 are superposed, the first container hole 3 and the fourth container hole 6 are symmetrically distributed relative to the horizontal central axis of the gasket 2, reagent bottles are respectively arranged in the first container hole 3, the second container hole 4, the third container hole 5 and the fourth container hole 6, P16 primary anti-concentration liquid, MCM Ki-67 primary anti-concentration liquid, MCM2 primary anti-concentration liquid and antibody dilution liquid are respectively filled in the reagent bottles, and the immune triple-staining detection of P16, 2 and Ki-67 tissue chemistry is scientific, efficient and stable, and can well meet requirements of science, stability, and the requirements of tissue chemistry, The need for work;

a fifth container hole 7 is distributed on the right side of the first container hole 3, a sixth container hole 8 is distributed on one side of the fifth container hole 7, a seventh container hole 9 is distributed on one side of the sixth container hole 8, an eighth container hole 10 is distributed on one side of the seventh container hole 9, first aseptic device holes 11 are distributed on the right side of the eighth container hole 10, reagent bottles are respectively placed in the fifth container hole 7, the sixth container hole 8, the seventh container hole 9 and the eighth container hole 10, the fifth container hole 7 and the eighth container hole 10 are coincident with the horizontal central axis of the eighth container hole 10, the horizontal central axes of the liner 2 are symmetrically distributed about the horizontal central axis of the liner 2, goat anti-mouse working fluid, goat anti-rabbit working fluid, DAB staining substrate 9 and DAB staining substrate are respectively contained in the reagent bottles, and the immunohistochemical staining test of a user is facilitated, the immunohistochemical triple staining of P16, MCM2 and Ki-67, in the staining process, primary antibodies of two proteins are mixed and then the sample is incubated, and meanwhile, the expression of the two proteins is detected on a sample slide, so that the method is cheap and rapid on the premise of accurately screening the early cervical cancer, and the detection speed is almost doubled. The kit is superior to similar immunohistochemical single staining kit for detecting P16 gene or MCM2 and Ki-67 gene only. The results of the immunohistochemical triple staining methods of P16, MCM2 and Ki-67 are displayed on the same sample slide, namely the results are displayed on the same sample slide, so that the accuracy of pathological slide reading of experimenters is improved, the observation is convenient, the detection accuracy is improved, the possibility of false positive and false negative is reduced, and the possibility of misjudgment is reduced;

second sterile device holes 12 are distributed on the right side of the first sterile device hole 11, the horizontal central axes of the first sterile device hole 11 and the second sterile device hole 12 are parallel, the length and width of the first sterile device hole 11 are equal to the length and width of the second sterile device hole 12, and buffer solution reagent bottles arranged in the first sterile device hole 11 and the second sterile device hole 12 are large in size, so that the transverse lying design is adopted for saving space, the stress of the device is more reasonable, and the service life of the device is prolonged;

first aseptic device hole 11 one end is provided with first loose-leaf 13, and second aseptic device hole 12 one end is provided with second loose-leaf 14, is swing joint between first loose-leaf 13 and the first aseptic device hole 11, and the coincidence of the vertical axis in first loose-leaf 13 and first aseptic device hole 11, through this structure, the user need mention first loose-leaf 13, second loose-leaf 14 then take out the reagent bottle and use, and the user operation is more convenient.

The working principle is as follows: for the immunohistochemical detection kit of P16, MCM2 and Ki67, the vertical central axis of the box body 1 coincides with the vertical central axis of the liner 2, the structure is arranged, the kit is compact in structure, convenient to carry, good in repeatability, time-saving and labor-saving, the liner 2 can buffer the impact generated by the shaking of the device on the reagent bottle in the box body 1, the safety of the reagent bottle is ensured, and the requirements of a user are met, the length and width of the first sterile device hole 11 are equal to the length and width of the second sterile device hole 12, and as the volume of the buffer solution reagent bottle arranged in the first sterile device hole 11 and the second sterile device hole 12 is larger, in order to save space, the transverse lying design is adopted, the stress of the device is more reasonable, the service life of the device is prolonged, the first hinge 13 coincides with the vertical central axis of the first sterile device hole 11, and through the structure, the user needs to lift up the first hinge 13, The second loose-leaf 14 is then taken out of the reagent bottle for use, and the operation is more convenient for users.

The immunohistochemical staining detection method comprises seven steps, namely primary antibody incubation, secondary antibody incubation, DAB staining, red staining, counterstaining, mounting and result analysis.

Firstly, primary anti-incubation is carried out, after the kit is opened, a reagent bottle is taken out of a first container hole 3, a second container hole 4, a third container hole 5 and a fourth container hole 6, 1.85ml of antibody diluent and 100 mu l of p16 primary anti-concentrate are mixed and added into 100 mu l of MCM2 and Ki-67 primary anti-concentrate to prepare p16/l of MCM2 and Ki-67 primary anti-work mixture, a new label is pasted, the mixture is kept in a dark place at the temperature of 2-8 ℃ for later use, 50 mu l/piece of primary anti-work mixture is dripped, a wet box is incubated for 30min at the temperature of 37 ℃, PBST is rinsed for 3min multiplied by 3 times and is properly dried;

then, performing secondary antibody incubation, after the kit is opened, taking out a reagent bottle from a fifth container hole 7, a sixth container hole 8, a seventh container hole 9 and an eighth container hole 10, adding 1ml of goat-anti-mouse secondary antibody working solution into 1ml of goat-anti-rabbit secondary antibody working solution to prepare a goat-anti-mouse/rabbit-secondary antibody working mixed solution, pasting a new label, storing in a dark place at 2-8 ℃, for later use, dropwise adding 50 mul/piece of secondary antibody working mixed solution, incubating in a wet box at 37 ℃ for 15min, rinsing PBST for 3min multiplied by 3 times, and spin-drying moderately;

performing DAB staining, taking out buffer solution, preparing DAB working mixed solution (according to the sample amount, taking a proper amount of DAB staining substrate and DAB staining agent buffer solution, mixing uniformly), using immediately after preparation, using up within 4h, storing at room temperature (20-25 ℃) in the dark place, dripping 50 mu l/piece of DAB working mixed solution, incubating in a wet box at room temperature (20-25 ℃) for 5-10 min, washing with running water for 3min, and spin-drying moderately (the piece cannot be dried);

performing red dyeing to prepare red working mixed liquid, namely preparing the red working mixed liquid, namely using the red working mixed liquid, using the red working mixed liquid within 30min, storing the red working mixed liquid at room temperature (20-25 ℃) in a dark place, dripping 50 mu l/piece of red working mixed liquid, incubating the red working mixed liquid in a wet box at room temperature (20-25 ℃) for 10-30 min, flushing the red working mixed liquid for 3min by flowing water, and properly spin-drying the red working mixed liquid;

counterstaining is carried out, the cell slices are placed into hematoxylin dye for dip staining, the counterstaining is carried out for 1s at room temperature (20-25 ℃), and the washing is carried out for 3min by running water and the drying is carried out;

then sealing, dripping 1 drop of neutral quick sealing tablet, covering with cover glass, and evaluating under optical microscope

And finally, analyzing results according to the staining conditions of the cell nucleus and the cell cytoplasm of the same monolayer of cells or the same cell cluster. After P16 and MCM2 and Ki-67 antibodies are incubated, DAB and red dyes are subjected to triple staining, the cell nucleus of the same single-layer cell or cell cluster is blue or red, and the cytoplasm is not stained; or the cell nucleus is brown and the cell cytoplasm is brown; or the cell nucleus is red, the cytoplasm is not stained, namely a negative result is judged, after P16 and MCM2 and Ki-67 antibodies are incubated, the DAB dye is singly stained, the cell nucleus of a single-layer cell or a cell cluster is brown, and the cytoplasm is brown; and (3) singly dyeing the red dye, wherein the cell nucleus of a single-layer cell or a cell cluster is red, and the cytoplasm is not dyed, so that the positive result is judged.

The immunohistochemical triple staining of P16, MCM2 and Ki-67, in the staining process, primary antibodies of two proteins are mixed and then the sample is incubated, and meanwhile, the expression of the two proteins is detected on a sample slide, so that the method is cheap and rapid on the premise of accurately screening the early cervical cancer, and the detection speed is almost doubled. The kit is superior to similar immunohistochemical single staining kit for detecting P16 gene or MCM2 and Ki-67 gene only. The results of the immunohistochemical triple staining methods of P16, MCM2 and Ki-67 are displayed on the same sample slide, namely the results are displayed on the same sample slide, so that the accuracy of pathological slide reading of experimenters is improved, the observation is convenient, the detection accuracy is improved, the possibility of false positive and false negative is reduced, and the possibility of misjudgment is reduced.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

P16 and MCM2, Ki67 immunohistochemical detection kit comprising a cartridge body (1) and a first sterile device well (11), characterized in that: the inner wall of the box body (1) is attached with a liner (2), the edge of the upper surface of the liner (2) is provided with a first container hole (3), one side of the first container hole (3) is distributed with a second container hole (4), one side of the second container hole (4) is distributed with a third container hole (5), one side of the third container hole (5) is distributed with a fourth container hole (6), the right side of the first container hole (3) is distributed with a fifth container hole (7), one side of the fifth container hole (7) is distributed with a sixth container hole (8), one side of the sixth container hole (8) is distributed with a seventh container hole (9), one side of the seventh container hole (9) is distributed with an eighth container hole (10), the first sterile device hole (11) is distributed on the right side of the eighth container hole (10), and the right side of the first sterile device hole (11) is distributed with a second sterile device hole (12), one end of the first sterile device hole (11) is provided with a first hinge (13), and one end of the second sterile device hole (12) is provided with a second hinge (14).
2. The P16 and MCM2, Ki67 immunohistochemical detection kit of claim 1, characterized by: the inner wall of the box body (1) is attached to the outer wall of the gasket (2), and the vertical central axis of the box body (1) coincides with the vertical central axis of the gasket (2).
3. The P16 and MCM2, Ki67 immunohistochemical detection kit of claim 1, characterized by: the horizontal central axis of the first container hole (3), the second container hole (4), the third container hole (5) and the horizontal central axis of the fourth container hole (6) coincide, and the first container hole (3) and the fourth container hole (6) are symmetrically distributed about the horizontal central axis of the gasket (2).
4. The P16 and MCM2, Ki67 immunohistochemical detection kit of claim 1, characterized by: the horizontal central axis of the fifth container hole (7), the horizontal central axis of the sixth container hole (8), the horizontal central axis of the seventh container hole (9) and the horizontal central axis of the eighth container hole (10) coincide, and the fifth container hole (7) and the eighth container hole (10) are symmetrically distributed about the horizontal central axis of the gasket (2).
5. The P16 and MCM2, Ki67 immunohistochemical detection kit of claim 1, characterized by: the horizontal central axis of the first sterile device hole (11) is parallel to the horizontal central axis of the second sterile device hole (12), and the length and width of the first sterile device hole (11) are equal to the length and width of the second sterile device hole (12).
6. The P16 and MCM2, Ki67 immunohistochemical detection kit of claim 1, characterized by: the first hinge (13) is movably connected with the first sterile device hole (11), and the vertical central axis of the first hinge (13) is superposed with that of the first sterile device hole (11).