CN110501496A - A kind of immunocyte p16 detection kit - Google Patents
A kind of immunocyte p16 detection kit Download PDFInfo
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- CN110501496A CN110501496A CN201910803714.9A CN201910803714A CN110501496A CN 110501496 A CN110501496 A CN 110501496A CN 201910803714 A CN201910803714 A CN 201910803714A CN 110501496 A CN110501496 A CN 110501496A
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- immunocyte
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- G—PHYSICS
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- 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
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Abstract
The invention discloses a kind of immunocyte p16 detection kits, including fixer, blocking endogenous peroxydase, confining liquid, cleaning solution, first antibody, secondary antibody, DAB developing solution；The fixer is the paraformaldehyde of mass concentration 4%；The blocking endogenous peroxydase includes methanol and 30%H2O2, the methanol and 30%H2O2Volume ratio be 100:1；The confining liquid by weight, including 55 parts of PBS buffer solution, 5 parts of animal non-immune serum, 5 parts of sucrose, 5 parts of Tween-20,1 part of Proclin300,20 parts of skimmed milk power, 100 parts of distilled water；The cleaning solution includes the PBS buffer solution that pH is 7.2；The first antibody is goat-anti p16INK4aPolyclonal antibody；The secondary antibody is the goat anti-mouse igg antibody of horseradish peroxidase-labeled.The present invention can effectively improve the dyeing effect of immunocyte p16 detection.
Description
Technical field
The present invention relates to immunohistochemistry detection technique fields, and in particular to a kind of immunocyte p16 detection kit.
Background technique
Cervical carcinoma is one of most common malignant tumour of gynaecology, and disease incidence is only second to breast cancer and is in rejuvenation trend,
Seriously endanger body of women health.However, cervical carcinoma is unique clear cause of disease again, being expected to becomes first is swollen by what the mankind captured
Tumor.Therefore, prevention and early screening are of great significance to the morbidity and the death rate that reduce cervical carcinoma.
Currently, the main method of diagnosis of cervical cancer has: cervical exfoliated cell checks, human papilloma virus (HPV) detects,
Vaginoscopy, pathological biopsy etc..Cytology detection mainly carries out pathological staining to cervical exfoliated cell by pathologist
Inspections and examinations are carried out under microscope afterwards.This method operation is relatively simple, and patient's no pain is appropriate for routine screening.
The existing generally existing confining liquid of immunocyte p16 detection kit is perishable, unstable, holding time short lacks
It falls into, to seriously affect immunocyte p16 dyeing effect.
Summary of the invention
In view of the deficiencies of the prior art, the present invention is intended to provide a kind of immunocyte p16 detection kit, can effectively mention
The dyeing effect of high immunocyte p16 detection.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of immunocyte p16 detection kit, including fixer, blocking endogenous peroxydase, confining liquid, washing
Liquid, first antibody, secondary antibody, DAB developing solution；
The fixer is the paraformaldehyde of mass concentration 4%；
The blocking endogenous peroxydase includes methanol and 30%H2O2, the methanol and 30%H2O2Volume ratio
For 100:1；
The confining liquid by weight, including 55 parts of PBS buffer solution, 5 parts of animal non-immune serum, 5 parts of sucrose, is spat
Temperature -20 5 parts, 1 part of Proclin300,20 parts of skimmed milk power, 100 parts of distilled water；
The cleaning solution includes the PBS buffer solution that pH is 7.2；
The first antibody is goat-anti p16INK4aPolyclonal antibody；
The secondary antibody is the goat anti-mouse igg antibody of horseradish peroxidase-labeled.
Further, in the confining liquid, the mass concentration of the Tween-20 is 0.2%.
Further, in the confining liquid, the concentration of PBS buffer solution is 0.01~0.2mol/L.
Further, in the confining liquid, the mass concentration of the Proclin300 is 0.1%.
Further, in the confining liquid, the animal non-immune serum is the lowlenthal serum of mass concentration 5-15%.
It further, further include having NaCl in the cleaning solution, the mass concentration of NaCl is 0.85%-1%.
The beneficial effects of the present invention are:
In the confining liquid of kit of the invention, by increasing Proclin300, the stability of confining liquid can be enhanced, prevent
The nutritional ingredients such as stop object non-immune serum and skimmed milk power are rotten, so that the holding time of confining liquid is longer.And
Sucrose can play a protective role to animal non-immune serum and skimmed milk power, so that animal non-immune serum and skimmed milk power are more
Add stabilization, oxidation resistant, protection animal non-immune serum and skimmed milk power can also be played the role of in confining liquid ingress of air.
In addition, being used in combination by addition Tween-20 and animal non-immune serum, skimmed milk power, then it is non-to play cleaning in closing
Specific binding or the effect for combining unstable albumen, so that specific binding enhancing.
In addition, in the kit of the present embodiment, by adding NaCl in cleaning solution, sufficiently washing can be played and cut
Piece effectively reduces non-specific binding and reduces the effect of background stainings.
Specific embodiment
The invention will be further described below, it should be noted that the present embodiment premised on the technical program,
The detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to the present embodiment.
Embodiment 1
The present embodiment provides a kind of immunocyte p16 detection kit, the kit includes fixer, blocks endogenous
Peroxidase, confining liquid, cleaning solution, first antibody, secondary antibody, DAB developing solution；
The fixer is the paraformaldehyde of mass concentration 4%；
The blocking endogenous peroxydase includes methanol and 30%H2O2, the methanol and 30%H2O2Volume ratio
For 100:1；
The confining liquid by weight, including 55 parts of PBS buffer solution, 5 parts of animal non-immune serum, 5 parts of sucrose, is spat
Temperature -20 5 parts, 1 part of Proclin300,20 parts of skimmed milk power, 100 parts of distilled water；
The cleaning solution includes the PBS buffer solution that pH is 7.2；
The first antibody is goat-anti p16INK4aPolyclonal antibody；
The secondary antibody is the goat anti-mouse igg antibody of horseradish peroxidase-labeled.
Further, in the confining liquid, the mass concentration of the Tween-20 is 0.2%.
Further, in the confining liquid, the concentration of PBS buffer solution is 0.01~0.2mol/L.
Further, in the confining liquid, the mass concentration of the Proclin300 is 0.1%.
Further, the animal non-immune serum is the lowlenthal serum of mass concentration 5-15%.
It further, further include having NaCl in the cleaning solution, the mass concentration of NaCl is 0.85%-1%.
In the confining liquid of mentioned reagent box, by increasing Proclin300, the stability of confining liquid can be enhanced, prevent
The nutritional ingredients such as animal non-immune serum and skimmed milk power are rotten, so that the holding time of confining liquid is longer.And sugarcane
Sugar can play a protective role to animal non-immune serum and skimmed milk power, so that animal non-immune serum and skimmed milk power are more
Stablize, oxidation resistant, protection animal non-immune serum and skimmed milk power can also be played the role of in confining liquid ingress of air.Separately
Outside, it is used in combination by addition Tween-20 and animal non-immune serum, skimmed milk power, then can play the non-spy of cleaning in closing
The opposite sex combines or combines the effect of unstable albumen, so that specific binding enhancing.
In addition, in the kit of the present embodiment, by adding NaCl in cleaning solution, sufficiently washing can be played and cut
Piece effectively reduces non-specific binding and reduces the effect of background stainings.
Embodiment 2
The present embodiment provides the preparation methods of confining liquid described in embodiment 1, include the following steps:
S1, first with 1/3 distilled water dissolve skimmed milk power；
S2, again with 1/3 distilled water dissolving saccharose；
S3, will be after PBS buffer solution, animal non-immune serum, dissolved sucrose, Tween-20, Proclin300, dissolution
Skimmed milk power and remaining distilled water be mixed, and adjust pH to 7.2-7.8, obtain the confining liquid.
Embodiment 3
The present embodiment provides the application methods of kit described in embodiment 1, include the following steps:
S1, cervical cell sample slice is fixed into 10min with fixer, then dried；
S2, at room temperature will through step S1 treated slice be put into block intrinsic oversxidase in 20 minutes, then
Washing；
S3, it is washed with cleaning solution sample 3 times, each 1 minute every time；
After S4, cleaning to through step S3 treated slice be added dropwise confining liquid, close 30 minutes, get rid of supernatant；
S5, be incubated for box in, to through step S4 treated slice be added dropwise first antibody, be incubated for 60 minutes at 37 DEG C；
Then it is washed 3 times with cleaning solution；
S6, secondary antibody is instilled to the slice handled through step S5, is incubated for 60 minutes at 37 DEG C；Then it is washed with cleaning solution
3 times；
S7, DAB developing solution is added dropwise to the slice handled through step S6, develops the color 3-10 minutes, controlled under mirror；
S8, washing color development stopping；
S9, hematoxylin light dye nucleus 1-2 minutes；
S10, washing, oil blackeite；
S11, graded ethanol dehydration, dimethylbenzene is transparent, neutral gum sealing.
For those skilled in the art, it can be provided various corresponding according to above technical solution and design
Change and modification, and all these change and modification, should be construed as being included within the scope of protection of the claims of the present invention.
Claims (6)
1. a kind of immunocyte p16 detection kit, which is characterized in that including fixer, block endogenous peroxydase, envelope
Close liquid, cleaning solution, first antibody, secondary antibody, DAB developing solution；
The fixer is the paraformaldehyde of mass concentration 4%；
The blocking endogenous peroxydase includes methanol and 30%H2O2, the methanol and 30%H2O2Volume ratio be 100:
1；
The confining liquid by weight, including 55 parts of PBS buffer solution, 5 parts of animal non-immune serum, 5 parts of sucrose, Tween-20
5 parts, 1 part of Proclin300,20 parts of skimmed milk power, 100 parts of distilled water；
The cleaning solution includes the PBS buffer solution that pH is 7.2；
The first antibody is goat-anti p16INK4aPolyclonal antibody；
The secondary antibody is the goat anti-mouse igg antibody of horseradish peroxidase-labeled.
2. immunocyte p16 detection kit according to claim 1, which is characterized in that described to spit in the confining liquid
The mass concentration of temperature -20 is 0.2%.
3. immunocyte p16 detection kit according to claim 1, which is characterized in that in the confining liquid, PBS is slow
The concentration of fliud flushing is 0.01~0.2mol/L.
4. immunocyte p16 detection kit according to claim 1, which is characterized in that described in the confining liquid
The mass concentration of Proclin300 is 0.1%.
5. immunocyte p16 detection kit according to claim 1, which is characterized in that described dynamic in the confining liquid
Object non-immune serum is the lowlenthal serum of mass concentration 5-15%.
6. immunocyte p16 detection kit according to claim 1, which is characterized in that further include in the cleaning solution
There is NaCl, the mass concentration of NaCl is 0.85%-1%.
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| CN201910803714.9A CN110501496A (en) | 2019-08-28 | 2019-08-28 | A kind of immunocyte p16 detection kit |
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| CN201910803714.9A CN110501496A (en) | 2019-08-28 | 2019-08-28 | A kind of immunocyte p16 detection kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112180090A (en) * | 2020-10-09 | 2021-01-05 | 深圳市森盈生物科技有限公司 | Kit of monoclonal antibody specifically bound by immune cell p16 |
| CN115901401A (en) * | 2023-01-09 | 2023-04-04 | 深圳市森盈生物科技有限公司 | P16 immunocytochemistry staining kit and staining method |
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| CN107202888A (en) * | 2017-07-11 | 2017-09-26 | 广州江元医疗科技有限公司 | A kind of p16 for cervical liquid-based cellsINK4aImmune labeled colour reagent box |
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Patent Citations (9)
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| CN101940916A (en) * | 2009-07-03 | 2011-01-12 | 湖北工业大学 | Konjac glucomannan stroma protein molecularly imprinted membrane as well as preparation method and application thereof |
| CN102621301A (en) * | 2011-01-28 | 2012-08-01 | 上海铭源数康生物芯片有限公司 | Preparation method of nitrocellulose membrane-based protein chip blocking liquid |
| CN102207502A (en) * | 2011-03-25 | 2011-10-05 | 宁波大学 | Mercury ion test paper and preparation method thereof |
| CN102618502A (en) * | 2012-01-09 | 2012-08-01 | 浙江大学 | Hybridoma cell strain capable of secreting monoclonal antibodies to quinolones and application of monoclonal antibodies thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112180090A (en) * | 2020-10-09 | 2021-01-05 | 深圳市森盈生物科技有限公司 | Kit of monoclonal antibody specifically bound by immune cell p16 |
| CN115901401A (en) * | 2023-01-09 | 2023-04-04 | 深圳市森盈生物科技有限公司 | P16 immunocytochemistry staining kit and staining method |
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Application publication date: 20191126 |