CN112881691A - Immunocytochemistry labeling developing kit for cervical cancer auxiliary diagnosis - Google Patents
Immunocytochemistry labeling developing kit for cervical cancer auxiliary diagnosis Download PDFInfo
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57411—Specifically defined cancers of cervix
Abstract
The invention relates to the technical field of cell color reagent, in particular to an immunocytochemistry labeling color reagent kit for cervical cancer auxiliary diagnosis, which comprises ready-to-use antibody working solution, reagent C solution, reagent D solution, reagent E solution, reagent F solution and reagent G solution; ready-to-use antibody working solution: the kit comprises an antibody reagent, bovine serum albumin, non-reducing sugar, fish skin gelatin, a preservative proclin-950, green pigment of tender leaves, polyethylene glycol, casein, a surfactant, 4-aminoantipyrine and sodium chloride, wherein the antibody reagent comprises a reagent A solution and a reagent B solution. The invention is innovative in blue returning mode, has better blue returning effect than other manufacturers in the market, is simpler in mounting mode, and greatly shortens the experimental time.
Description
Technical Field
The invention relates to the technical field of cell color reagent, in particular to an immunocytochemistry labeling color development kit for cervical cancer auxiliary diagnosis.
Background
At present, patent document CN201510085334.8 proposes that cervical epithelial tissue specimens are subjected to immunohistochemical double staining to determine whether cervical epithelium has neoplastic lesions. The method is used for judging whether high-degree neoplastic lesions (CIN III) occur or not by using an immunoenzyme substrate chromogenic method for two markers of phosphoprotein (stathmin) and mini-chromosome maintenance protein (MCM 2) of a cervical squamous epithelial tissue sample. However, in the method, the sample is obtained by cervical biopsy, which belongs to invasive detection, on one hand, the individual is injured, and on the other hand, the patient needs to be sampled secondarily according to the current cervical cancer screening standard, so that the sampling difficulty is increased; also, the diagnostic studies of the two markers of this approach in precancerous lesions are more limited and the clinical application is more limited.
Chinese patent CN201210567622.3 proposes to detect P16INK4a protein expression by immunocytochemistry method with cervical exfoliated cells to determine whether there is a pre-cervical cancer lesion, but P16INK4a protein is over-expressed not only in abnormally proliferating cells of cervix, but also in metaplastic cells of oviduct, endometrial cells and normal columnar cells, and the final diagnosis still needs to be determined by combining cell morphology, so that the workload of doctors is relatively limited.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide an immunocytochemistry marker chromogenic kit for auxiliary diagnosis of cervical cancer.
In order to achieve the purpose, the invention provides the following technical scheme: an immunocytochemistry labeling color development kit for cervical cancer auxiliary diagnosis comprises ready-to-use antibody working solution, reagent C solution, reagent D solution, reagent E solution, reagent F solution and reagent G solution;
ready-to-use antibody working solution: comprises a reagent solution A and a reagent solution B. The components comprise antibody reagent, bovine serum albumin, non-reducing sugar, fish skin gelatin, preservative proclin-950, green pigment of tender leaves, polyethylene glycol, casein, surfactant, 4-aminoantipyrine and sodium chloride;
reagent A solution: a mixed primary antibody formed by combining the P16INK4a antibody and the Ki-67 antibody;
reagent B solution: the HRP-labeled goat anti-mouse polymer coupled secondary antibody and the AP-labeled goat anti-rabbit polymer coupled secondary antibody are combined to form a mixed secondary antibody;
reagent C solution: a blocking agent;
reagent D solution: diaminobenzidine;
reagent E solution: a buffer solution for developing the color of the reagent D solution;
reagent F solution: fixing the nucleus to the chromogen;
reagent G solution: and a buffer for developing the color of the reagent F.
Preferably, the reagent A solution is a Tris buffer solution of the p16INK4a monoclonal antibody and the Ki-67 monoclonal antibody.
Preferably, the pH value of the Tris buffer solution is 7.19-7.81, wherein the concentration of the p16INK4a antibody is 0.09-1.5 mg/L; the concentration of the Ki-67 antibody is 0.09-1.5 mg/L.
Preferably, the mixed secondary antibody formed by combining the goat anti-mouse polymer conjugate secondary antibody marked by the HRP in the reagent B solution is prepared by dissolving polylysine in a phosphate buffer solution, adding a proper amount of sodium periodate for oxidation, dialyzing overnight by using the same phosphate buffer solution as a polylysine matrix, adding a proper amount of sodium periodate for oxidation by using water-soluble horseradish peroxidase, and dialyzing overnight by using a glycol termination reaction; and (3) mixing oxidized polylysine, a goat anti-mouse antibody and activated horseradish peroxidase, reacting, dialyzing overnight, and diluting to working solution by using a preservation solution.
Preferably, the AP-labeled goat anti-rabbit polymer conjugated secondary antibody in the reagent B solution is prepared by dissolving polylysine in phosphate buffer solution, adding appropriate amount of sodium periodate for oxidation, dialyzing with the same phosphate buffer solution as polylysine matrix overnight, adding appropriate amount of sodium periodate for oxidation in water-soluble alkaline phosphatase, and dialyzing overnight by using glycol termination reaction; and (3) mixing oxidized polylysine, a goat anti-rabbit ki67 antibody and activated horseradish peroxidase, reacting, dialyzing overnight, and diluting to a working solution by using a preservation solution.
Preferably, the pH value of the phosphate buffer solution of the polylysine matrix is 5-9.
Preferably, the concentration of the bovine serum albumin is 0.10-2.65 g/L; the concentration of the non-reducing sugar is 10-20 g/L; the concentration of the fish skin gelatin is 1-4 g/L; the concentration of the preservative is 0.2-5 ml/L; the green pigment concentration of the tender leaves is 0.1-0.5 g/L; the concentration of the polyethylene glycol is 10-50 g/L; the casein concentration is 0.1g-1 g/L; the concentration of the 4-aminoantipyrine is 0.1g to 1.5 g/L; the concentration of the sodium chloride is 10g-30 g/L; the content of the surfactant is 0.005-1.2 wt%.
Preferably, the blocking agent is hydrogen peroxide solution with the concentration of 1% -5.1%.
Preferably, the reagent solution A, the reagent solution B, the reagent solution C, the reagent solution D, the reagent solution E, the reagent solution F and the reagent solution G are all stored in an environment with the temperature of 2-8 ℃.
In order to achieve the above purpose, the invention also provides the following technical scheme: an application method of an immunocytochemical labeling chromogenic kit for cervical cancer auxiliary diagnosis comprises the following steps:
the S1 kit is taken out from the refrigeration environment, placed at room temperature for 15-30 min and then used;
s2 white slice preparation:
(1) placing the cell preservation solution bottle containing the sample on a sample oscillator for oscillation for not less than 1 min;
(2) adding a diluent into the tabletting bin;
(3) adding a specimen into the film making bin and then standing;
(4) pouring out residual cell liquid in the slide making bin, unscrewing the slide making bin, taking out the slide, flushing the slide with running water, inserting the slide into a slide rack, putting the slide rack into 95% ethanol for fixation, taking out the slide rack, and standing at room temperature for 30-60 min;
s3 antigen retrieval: putting the white slices into PBST for hydration, putting a proper amount of antigen repairing liquid into a non-magnetic cup, adding tap water into a pressure cooker to 1/4-1/3 of a container, then putting the non-magnetic cup filled with the repairing liquid into the cooker, covering a cooker cover and a cup cover, adjusting an electromagnetic oven to 2100w for heating for 7min, adjusting the electromagnetic oven to 300w, opening the cooker cover and the cup cover to add the hydrated white slices, covering the cooker cover, setting a countdown timer for 20min, opening the cover after the timing is finished, naturally cooling the cover until the repairing liquid does not scald hands, washing the white slices with pure water for three times, taking out the white slices, sequentially drawing circles, and washing the white slices with PBST for 3 times after the circles are drawn, wherein each time is 2 min;
s4 blocking: throwing off the PBST on the white slices, dripping one drop of blocking agent on each slice, incubating for 10min at room temperature, and washing the PBST for 3 times and 2min each time after the incubation is finished;
s5 primary antibody incubation: throwing off PBST on the white tablets, adding one drop of primary antibody to each white tablet, incubating at room temperature for 35min, and washing PBST for 3 times, each time for 2 min;
s6 secondary antibody incubation: throwing off PBST on the slices, adding a drop of secondary antibody to each white slice, incubating at room temperature for 20min, and washing PBST for 3 times, each time for 2 min;
s7 Fast Red color development: throwing off PBST on the white slices, adding 60ul Fast Red color developing agent into each white slice, incubating at room temperature for 10min, and washing PBST for 3 times, each time for 2 min;
s8 addition of enzyme: adding horseradish peroxidase into each hole except the blank hole by 50 mu L;
s9 DAB color development: throwing off PBST on the white tablets, adding 60ul of DAB color developing agent into each white tablet, incubating at room temperature for 5min, and washing PBST for 3 times, each time for 2 min;
s10 counterstaining with hematoxylin and returning blue, namely, throwing off PBST on the white slices, dripping 4-6 drops of hematoxylin each piece, incubating at room temperature for 1-5min, washing residual hematoxylin with pure water, putting into a container filled with EDTA, returning blue for 3min, and putting into pure water after the blue returning is finished;
s11 wet seal and microscopic examination: taking the white slice out of the pure water, adding a drop of aqueous mounting agent on the wet slice for mounting, naturally airing or drying in an oven, and then diagnosing and interpreting under a microscope of a diagnostician;
s12 interpretation guide: the detection interpretation is independent of cell morphology, the cytoplasm is stained brown, the nucleus is stained red, a P16 brown signal and a Ki-67 red signal are positioned in the same cell together, the cell is positive, and the nucleus brown is P16 single positive; the nucleus is red, and the cytoplasm is light blue, so that the ki67 is single positive; blue nuclei were double negative.
Compared with the prior art, the invention has the beneficial effects that: the kit takes polylysine as a supporting framework, and the composite antibody and the signal marker form the lysine polyclonal antibody, which carries a large amount of signal markers, greatly improves the signal intensity, reduces the use concentration of the antibody, and avoids non-specific signal interference. Meanwhile, the macromolecular compound is combined with the enzyme at multiple sites, so that stable support is provided for the natural three-dimensional conformation of the enzyme, the stability of the enzyme and the antibody is greatly maintained, and efficient bonding between macromolecules is realized. The kit greatly reduces the dosage of the antibody and the operation flow and time. The blue returning mode is original, the mounting method is simpler, and the time is greatly shorter than Roche.
Drawings
FIG. 1 is a microscopic view of the cell staining of the present invention;
FIG. 2 is a graph showing the staining of the nucleus and cytoplasm of P16in a single positive state according to the present invention;
FIG. 3 is a nuclear staining map of ki67 with single positive status according to the present invention;
FIG. 4 is a graph showing the staining of negative cells according to the present invention;
FIG. 5 is a graph of tissue staining according to the present invention.
Detailed Description
The following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: an immunocytochemistry labeling color development kit for cervical cancer auxiliary diagnosis comprises ready-to-use antibody working solution, reagent C solution, reagent D solution, reagent E solution, reagent F solution and reagent G solution;
ready-to-use antibody working solution: comprises a reagent solution A and a reagent solution B. The components are antibody reagent, bovine serum albumin, non-reducing sugar, fish skin gelatin, preservative proclin-950, green pigment of tender leaves, polyethylene glycol, casein, surfactant, 4-aminoantipyrine and sodium chloride;
reagent A solution: a mixed primary antibody formed by combining the P16INK4a antibody and the Ki-67 antibody;
reagent B solution: the HRP-labeled goat anti-mouse polymer coupled secondary antibody and the AP-labeled goat anti-rabbit polymer coupled secondary antibody are combined to form a mixed secondary antibody;
reagent C solution: a blocking agent;
reagent D solution: diaminobenzidine;
reagent E solution: a buffer solution for developing the color of the reagent D solution;
reagent F solution: fixing the nucleus to the chromogen;
reagent G solution: and a buffer for developing the color of the reagent F.
The reagent A solution is a Tris buffer solution of a p16INK4a monoclonal antibody and a Ki-67 monoclonal antibody.
The pH value of the Tris buffer solution is 7.19-7.81, wherein the concentration of the p16INK4a antibody is 0.09-1.5 mg/L; the concentration of the Ki-67 antibody is 0.09-1.5 mg/L.
The mixed secondary antibody formed by combining the goat anti-mouse polymer conjugate secondary antibody marked by the HRP in the reagent B solution is prepared by dissolving polylysine in phosphate buffer solution, adding appropriate sodium periodate for oxidation, dialyzing overnight by using the phosphate buffer solution with the same polylysine matrix, adding appropriate sodium periodate for oxidation by using water-soluble horseradish peroxidase, and dialyzing overnight by using glycol to terminate the reaction; oxidized polylysine, a goat anti-mouse p16INK4a antibody and activated horseradish peroxidase are mixed and reacted, dialyzed overnight, and diluted to working solution by using a preservation solution.
Dissolving polylysine in phosphate buffer solution, adding appropriate amount of sodium periodate for oxidation, dialyzing with the same phosphate buffer solution as polylysine matrix overnight, adding appropriate amount of sodium periodate for oxidation in water-soluble alkaline phosphatase, terminating reaction with ethylene glycol, and dialyzing overnight; and (3) mixing oxidized polylysine, a goat anti-rabbit ki67 antibody and activated horseradish peroxidase, reacting, dialyzing overnight, and diluting to a working solution by using a preservation solution.
The pH values of the phosphate buffer solutions of the polylysine matrixes are 5-9.
The concentration of the bovine serum albumin is 0.10-2.65 g/L; the concentration of the non-reducing sugar is 10-20 g/L; the concentration of the fish skin gelatin is 1-4 g/L; the concentration of the preservative is 0.2-5 ml/L; the green pigment concentration of the tender leaves is 0.1-0.5 g/L; the concentration of the polyethylene glycol is 10-50 g/L; the casein concentration is 0.1g-1 g/L; the concentration of the 4-aminoantipyrine is 0.1g to 1.5 g/L; the concentration of the sodium chloride is 10g-30 g/L; the content of the surfactant is 0.005-1.2 wt%.
The blocking agent is hydrogen peroxide solution with the concentration of 1% -5.1%.
And the reagent solution A, the reagent solution B, the reagent solution C, the reagent solution D, the reagent solution E, the reagent solution F and the reagent solution G are stored in an environment at the temperature of 2-8 ℃.
Based on the above, in the kit of the present invention,
the reagent A solution is a mixed primary antibody formed by combining 1.5-6.4ml of P16INK4a antibody and Ki-67 antibody;
the reagent B solution is a mixed secondary antibody formed by combining 1.5-6.4ml of HRP-marked goat-anti-mouse polymer coupled secondary antibody and AP-marked goat-anti-rabbit polymer coupled secondary antibody;
the reagent C solution is 1.5-6.4ml of blocking agent;
the reagent D solution is 0.1ml to 0.44ml of diaminobenzidine (DAB concentrated color solution);
the reagent E solution is 2-8.8ml of buffer solution (DAB substrate buffer solution) for developing the color of the reagent D solution;
the reagent F solution is 0.03ml to 0.132ml of nuclear Fast Red chromogen (Fast Red concentrated developing solution);
the reagent G solution was 2 to 8.2ml of a buffer solution (Fast Red substrate buffer solution) for developing the color of the reagent F solution.
An application method of an immunocytochemical labeling chromogenic kit for cervical cancer auxiliary diagnosis comprises the following steps:
the S1 kit is taken out from the refrigeration environment, placed at room temperature for 15-30 min and then used;
s2 white slice preparation:
(1) placing the cell preservation solution bottle containing the sample on a sample oscillator for oscillation for not less than 1 min;
(2) adding 3mL of diluent into the tabletting bin;
(3) adding 2.5mL of specimen into the film making bin, and standing for 50 min;
(4) pouring out residual cell liquid in the slide making bin, unscrewing the slide making bin to take out the slide, slightly washing the slide with small flow of water, inserting the slide into a slide rack, putting the slide rack into fresh 95% ethanol for fixing for 30min, then taking out the slide rack, and standing at room temperature for 30-60min (so that the alcohol on the slide is completely volatilized);
s3 antigen repair (water barrier repair): putting the white chips into PBST, hydrating for 5min, putting a proper amount (about 600ml) of antigen repairing liquid into a non-magnetic cup, adding tap water into a pressure cooker from 1/4-1/3 of a container, then putting the non-magnetic cup filled with the repairing liquid into the pressure cooker, covering a cooker cover and a cup cover, heating at 2100w for 7min, adjusting an induction cooker to 300w, opening the cooker cover and the cup cover, adding the hydrated white chips, covering the cover, setting a countdown timer for 20min on the induction cooker, and after the timer is finished, opening the cover and naturally cooling until the repairing liquid does not scald hands. Washing the white film with pure water for three times, taking out the white film, sequentially circling, and washing with PBST for 3 times, each time for 2 min;
s4 blocking: throwing off the PBST on the white slices, dripping one drop of blocking agent on each slice, incubating for 10min at room temperature, and washing the PBST for 3 times and 2min each time after the incubation is finished;
s5 primary antibody incubation: throwing off PBST on the white tablets, adding one drop of primary antibody to each white tablet, incubating at room temperature for 35min, and washing PBST for 3 times, each time for 2 min;
s6 secondary antibody incubation: the PBST on the sections was spun off, a drop of secondary antibody was added to each white section, incubated at room temperature for 20min, and PBST was washed 3 times for 2min each.
S7 Fast Red color development: the PBST on the white slices was thrown away, 60ul Fast Red developer was added to each white slice, incubation was carried out at room temperature for 10min, PBST was washed 3 times, 2min each time.
S8 addition of enzyme: adding horseradish peroxidase 50 μ L to each well except for blank wells (for Fast Red preparation: Fast Red concentrated developing solution and Fast Red substrate buffer solution are prepared according to the ratio of 1: 74);
s9 DAB color development: and (3) throwing off the PBST on the white tablets, adding 60ul of DAB color developing agent into each white tablet, incubating at room temperature for 5min, and washing the PBST for 3 times for 2min each time. (Note: DAB Ready-to-use: DAB concentrated color solution and DAB substrate buffer solution were prepared in a ratio of 1:19
S10 counterstaining with hematoxylin and turning blue, throwing off PBST on the white slices, dripping 4-6 drops of hematoxylin each piece, incubating at room temperature for 1-5min, washing residual hematoxylin with pure water, placing in a container containing EDTA, turning blue for 3min, and placing in pure water after turning blue.
S11 wet seal and microscopic examination: and taking the white slice out of the pure water, adding a drop of aqueous mounting medium on the wet slice for mounting, naturally airing or drying in an oven, and then diagnosing under a microscope by a diagnostician. (note: the mounting agent can be put into a 45 ℃ oven for preheating in the tabletting stage, and the white pieces in the mounting need to be taken out from the water one by one, so that the pieces cannot be dried).
S12 interpretation guide: the assay was independent of cell morphology, with cytoplasmic staining brown (P16), nuclear staining red (Ki-67), P16 signal (brown) and Ki-67 signal (red) co-localized in the same cell, positive (FIG. 3), nuclear brown P16 single positive (FIG. 2), and nuclear brown P16 single positive. The nucleus was red and the cytoplasm was bluish, which was a single positive for ki67 (FIG. 4). Blue nuclei were double negative. The tissue staining is shown in FIG. 5.
Based on the method, after the antigen is incubated by a primary anti-reagent (a mixture of a mouse-derived p16 monoclonal antibody and a rabbit-derived Ki-67 monoclonal antibody), an antigen-antibody complex of a primary antibody and a target antigen is formed, a primary antibody in the antigen-antibody complex is incubated and combined with a secondary antibody reagent (a mixture of a HRP-labeled goat-anti-mouse polymer secondary antibody and an AP-labeled goat-anti-rabbit polymer secondary antibody), an antigen-antibody-secondary antibody complex is further formed, finally, a DAB color developing solution is catalyzed by HRP to form a brown deposit on a p16 antigen site, a Fast Red developing solution is catalyzed by AP to generate a Red deposit on a Ki-67 antigen site, and the staining result is evaluated by observation through an optical microscope.
By adopting the technical scheme of the kit and the using method, the instant reagent protected by multiple components is adopted, so that the time is saved, the operation is convenient, the stability of the active ingredients is improved, the active ingredients in the instant reagent can be stably stored for a long time, and the waste is avoided by adding more or less drops. The cervical exfoliated cell white sheet is subjected to antigen retrieval treatment and then incubated with a primary antibody reagent to form an antigen-antibody complex of a primary antibody and a target antigen, the primary antibody molecule in the antigen-antibody complex is incubated and combined with a secondary antibody reagent (a mixture of a HRP-labeled goat-anti-mouse polymer secondary antibody and an AP-labeled goat-anti-rabbit polymer secondary antibody) to further form an antigen-antibody-secondary antibody complex, finally, a DAB color developing solution is catalyzed by HRP to form a brown sediment on a p16 antigen locus, and the AP catalyzes a Fast Red color developing solution to generate a Red sediment on a Ki-67 antigen locus. The positioning effect and the color development effect are good, and the mark is more obvious.
Meanwhile, polylysine is used as a supporting framework, the composite antibody and the signal marker form a lysine polyclonal antibody, the lysine polyclonal antibody carries a large amount of signal markers, the signal intensity is greatly improved, the use concentration of the antibody is reduced, and non-specific signal interference is avoided. Meanwhile, the macromolecular compound is combined with the enzyme at multiple sites, so that stable support is provided for the natural three-dimensional conformation of the enzyme, the stability of the enzyme and the antibody is greatly maintained, and efficient bonding between macromolecules is realized. The kit greatly reduces the dosage of the antibody and the operation flow and time. The blue returning mode is original, the mounting method is simpler, and the time is greatly shorter than Roche. The invention has higher color rendering efficiency and detection sensitivity. The invention can be used for cervical decellularization and can also be used for staining tissues. The invention is innovative in blue returning mode, has better blue returning effect than other manufacturers in the market, is simpler in mounting mode, and greatly shortens the experimental time.
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 (10)
1. An immunocytochemistry labeled color development kit for cervical cancer auxiliary diagnosis, which is characterized in that: comprises ready-to-use antibody working solution, reagent C solution, reagent D solution, reagent E solution, reagent F solution and reagent G solution;
ready-to-use antibody working solution: comprises reagent A liquid and reagent B liquid. The ingredients include bovine serum albumin, non-reducing sugar, fish skin gelatin, preservative proclin-950, green pigment of tender leaf, polyethylene glycol, casein, surfactant, 4-aminoantipyrine and sodium chloride;
reagent A solution: a mixed primary antibody formed by combining the P16INK4a antibody and the Ki-67 antibody;
reagent B solution: the HRP-labeled goat anti-mouse polymer coupled secondary antibody and the AP-labeled goat anti-rabbit polymer coupled secondary antibody are combined to form a mixed secondary antibody;
reagent C solution: a blocking agent;
reagent D solution: diaminobenzidine;
reagent E solution: a buffer solution for developing the color of the reagent D solution;
reagent F solution: fixing the nucleus to the chromogen;
reagent G solution: and a buffer for developing the color of the reagent F.
2. The immunocytochemical labeling chromogenic kit for auxiliary diagnosis of cervical cancer according to claim 1, characterized in that: the reagent A solution is a Tris buffer solution of a p16INK4a monoclonal antibody and a Ki-67 monoclonal antibody.
3. The immunocytochemical labeling chromogenic kit for auxiliary diagnosis of cervical cancer according to claim 1, characterized in that: the pH value of the Tris buffer solution is 7.19-7.81, wherein the concentration of the p16INK4a antibody is 0.09-1.5 mg/L; the concentration of the Ki-67 antibody is 0.09-1.5 mg/L.
4. The immunocytochemical labeling chromogenic kit for auxiliary diagnosis of cervical cancer according to claim 1, characterized in that: the goat anti-mouse polymer coupled secondary antibody marked by HRP in the reagent B solution is prepared by dissolving polylysine in phosphate buffer solution, adding appropriate amount of sodium periodate for oxidation, dialyzing with the same phosphate buffer solution as polylysine matrix overnight, adding appropriate amount of sodium periodate for oxidation by water-soluble horseradish peroxidase, terminating reaction by using glycol, and dialyzing overnight; and (3) mixing oxidized polylysine, a goat anti-mouse antibody and activated horseradish peroxidase, reacting, dialyzing overnight, and diluting to working solution by using a preservation solution.
5. The immunocytochemical labeling chromogenic kit for auxiliary diagnosis of cervical cancer according to claim 4, characterized in that: dissolving polylysine in phosphate buffer solution, adding appropriate amount of sodium periodate for oxidation, dialyzing with the same phosphate buffer solution as polylysine matrix overnight, adding appropriate amount of sodium periodate for oxidation in water-soluble alkaline phosphatase, terminating reaction with ethylene glycol, and dialyzing overnight; and (3) mixing oxidized polylysine, a goat anti-rabbit ki67 antibody and activated horseradish peroxidase, reacting, dialyzing overnight, and diluting to a working solution by using a preservation solution.
6. The immunocytochemical labeling chromogenic kit for auxiliary diagnosis of cervical cancer according to claim 5, characterized in that: the pH values of the phosphate buffer solutions of the polylysine matrixes are 5-9.
7. The immunocytochemical labeling chromogenic kit for auxiliary diagnosis of cervical cancer according to claim 1, characterized in that: the concentration of the bovine serum albumin is 0.10-2.65 g/L; the concentration of the non-reducing sugar is 10-20 g/L; the concentration of the fish skin gelatin is 1-4 g/L; the concentration of the preservative is 0.2-5 ml/L; the green pigment concentration of the tender leaves is 0.1-0.5 g/L; the concentration of the polyethylene glycol is 10-50 g/L; the casein concentration is 0.1g-1 g/L; the concentration of the 4-aminoantipyrine is 0.1g to 1.5 g/L; the concentration of the sodium chloride is 10g-30 g/L; the content of the surfactant is 0.005-1.2 wt%.
8. The immunocytochemical labeling chromogenic kit for auxiliary diagnosis of cervical cancer according to claim 1, characterized in that: the blocking agent is hydrogen peroxide solution with the concentration of 1% -5.1%.
9. The immunocytochemical labeling chromogenic kit for auxiliary diagnosis of cervical cancer according to claim 1, characterized in that: and the reagent solution A, the reagent solution B, the reagent solution C, the reagent solution D, the reagent solution E, the reagent solution F and the reagent solution G are stored in an environment at the temperature of 2-8 ℃.
10. The use method of the immunocytochemistry labeling color development kit for the auxiliary diagnosis of the cervical cancer is characterized in that: the method comprises the following steps:
the S1 kit is taken out from the refrigeration environment, placed at room temperature for 15-30 min and then used;
s2 white slice preparation:
(1) placing the cell preservation solution bottle containing the sample on a sample oscillator for oscillation for not less than 1 min;
(2) adding a diluent into the tabletting bin;
(3) adding a specimen into the film making bin and then standing;
(4) pouring out residual cell liquid in the slide making bin, unscrewing the slide making bin, taking out the slide, flushing the slide with running water, inserting the slide into a slide rack, putting the slide rack into 95% ethanol for fixation, taking out the slide rack, and standing at room temperature for 30-60 min;
s3 antigen retrieval: putting the white slices into PBST for hydration, putting a proper amount of antigen repairing liquid into a non-magnetic cup, adding tap water into a pressure cooker to 1/4-1/3 of a container, then putting the non-magnetic cup filled with the repairing liquid into the cooker, covering a cooker cover and a cup cover, adjusting an electromagnetic oven to 2100w for heating for 7min, adjusting the electromagnetic oven to 300w, opening the cooker cover and the cup cover to add the hydrated white slices, covering the cooker cover, setting a countdown timer for 20min, opening the cover after the timing is finished, naturally cooling the cover until the repairing liquid does not scald hands, washing the white slices with pure water for three times, taking out the white slices, sequentially drawing circles, and washing the white slices with PBST for 3 times after the circles are drawn, wherein each time is 2 min;
s4 blocking: throwing off the PBST on the white slices, dripping one drop of blocking agent on each slice, incubating for 10min at room temperature, and washing the PBST for 3 times and 2min each time after the incubation is finished;
s5 primary antibody incubation: throwing off PBST on the white tablets, adding one drop of primary antibody to each white tablet, incubating at room temperature for 35min, and washing PBST for 3 times, each time for 2 min;
s6 secondary antibody incubation: throwing off PBST on the slices, adding a drop of secondary antibody to each white slice, incubating at room temperature for 20min, and washing PBST for 3 times, each time for 2 min;
s7 Fast Red color development: throwing off PBST on the white slices, adding 60ul Fast Red color developing agent into each white slice, incubating at room temperature for 10min, and washing PBST for 3 times, each time for 2 min;
s8 addition of enzyme: adding horseradish peroxidase into each hole except the blank hole by 50 mu L;
s9 DAB color development: throwing off PBST on the white tablets, adding 60ul of DAB color developing agent into each white tablet, incubating at room temperature for 5min, and washing PBST for 3 times, each time for 2 min;
s10 counterstaining with hematoxylin and turning blue, namely throwing off PBST on the white slices, dripping 4-6 drops of hematoxylin each piece, incubating at room temperature for 1-5min, washing residual hematoxylin with pure water, putting into a container filled with EDTA to turn blue for 3min, and putting into pure water after the blue turning is finished;
s11 wet seal and microscopic examination: taking the white slice out of the pure water, adding a drop of aqueous mounting agent on the wet slice for mounting, naturally airing or drying in an oven, and then diagnosing and interpreting under a microscope of a diagnostician;
s12 interpretation guide: the detection interpretation is independent of cell morphology, the cytoplasm is stained brown, the nucleus is stained red, a P16 brown signal and a Ki-67 red signal are positioned in the same cell together, the cell is positive, and the nucleus brown is P16 single positive; the nucleus is red, and the cytoplasm is light blue, so that the ki67 is single positive; blue nuclei were double negative.
Priority Applications (1)
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