CN206975044U - A kind of detection kit of people p16 albumen - Google Patents
A kind of detection kit of people p16 albumen Download PDFInfo
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- CN206975044U CN206975044U CN201720323879.2U CN201720323879U CN206975044U CN 206975044 U CN206975044 U CN 206975044U CN 201720323879 U CN201720323879 U CN 201720323879U CN 206975044 U CN206975044 U CN 206975044U
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Abstract
The utility model provides a kind of detection kit of people p16 albumen, the kit includes box body and the clamshell lid being connected with box body, it is characterized in that, there is the groove for placing reagent bottle and the reagent bottle on low groove in the box body, 8 reagent bottles and 1 powdered reagent groove are provided with the box, 8 reagent bottles are divided into three row from the left surface of box body to right flank, wherein, a row positioned at left surface include 3 reagent bottles, a row positioned at right flank include 3 reagent bottles, a row positioned at centre include 2 reagent bottles, the trailing flank that the powdered reagent groove is fixed in box body.Kit of the present utility model can quick detection people's p16 albumen immunohistochemical kit detection there is agent combination comprehensive, high sensitivity, high specificity, simple to operate, the time is quick.
Description
Technical field
The utility model belongs to medical instruments field, is related to a kind of detection kit of people p16 albumen.
Background technology
P16 tumor suppressor genes are located on human chromosomal 9p21 galianconism, belong to the member of INK4 class cell cycle inhibitors
One of.P16 albumen can combine cell cycle protein dependent kinase 4 (CDK4) and 6 (CDK6), and maintain retinoblastoma
Gene outcome pRb albumen is continuously in low phosphorylation high activity state.PRb can then combine E2F transcription factors and prevent the G1 phases from entering
Enter the cell cycle progression of S phases.Expression of the p16 albumen in senile cell increased.P16 protein overexpressions are in such case
Lower death and the apoptosis that can finally promote cell.In the related tumour of most of inhuman papillomavirus (HPV) infection, including
In colon cancer, mammary gland, pancreas, melanoma and the incidence cancer closely related with smoking, all there is missing, mutation in p16 genes
Or epigenetic silence and cause loss of function.Therefore, result of the p16 protein immunizations groupization detection in these tumours is typically
Negative.
And then completely contradicted in the related tumour of HPV infection.Host cell gene group is integrated into after HPV viruse infection
In, cause the generation of E6 and E7 oncoproteins.E6 can degrade and p53 and prevent Apoptosis.E7 then functionally inactivates pRb,
So as to prevent pRb combination E2F transcription factors.Key one rings of the pRb as positive feedback loop, causes p16 in nucleus in turn
With the expression quantity increase in cytoplasm, it as a result can be tested and detected by p16 protein immunizations groupization.It has recently been demonstrated that turn
The high HPV of record activity can cause very high-caliber p16 expression.The E7 albumen of low-risk HPV viruse coding is in some bioids
Learn and biological property in terms of be different from excessive risk HPV coding, result be infection low-risk HPV viruse cell in not
It was observed that p16 protein accumulations are expressed.In view of the result of the above, the p16 protein overexpressions detected by SABC can be made
For the sensitivity label of high-risk HPV infection in identification tumour.However, research shows that p16 is also expressed in many other cancers, such as
Embryonal-cell lipoma, sdenocarcinoma of stomach, Hodgkin lymphoma, NHL, adenocarcinoma of lung, neuroendocrine carcinoma and non-HPV infection machine
Uterine cancer caused by system etc..Gene delection, point mutation, function mutation or the imbalance of Rb paths cause pRb inactivation to be that these are non-
The main reason for HPV infection tumour high-expression p16 albumen.So expressed with immunohistochemistry technique detection p16 more than for diagnosis
Cancer has important reference significance.
Immunohistochemistry is the characteristic using antibody energy molecule of the antigen binding, then by antibody label it is aobvious
Color, come detect or position tissue or cell in certain protein material technology.But conventional immunohistochemistry skill
Time-consuming for art experiment, and whole experiment at least needs a few hours to complete, and causing can not with immunohistochemistry technique detection p16 expression
In time, result is quickly and efficiently obtained, it is hindered and diagnoses the clinical practice of cancer.
At present, the quick detection kit of people p16 albumen is there is not yet Patents, preparing one can be to people's p16 egg
The kit being used for quickly detecting in vain is just particularly important.
Utility model content
The purpose of this utility model is to provide a kind of detection kit of people p16 albumen, and the kit detection time is short,
And accurate testing result can be provided.
To reach this purpose of utility model, the utility model uses following technical scheme：
On the one hand, the utility model provides a kind of detection kit of people p16 albumen, including box body and is connected with box body
Clamshell lid, there is in the box body groove for placing reagent bottle and the reagent bottle on low groove, set in the box
There are 8 reagent bottles and 1 powdered reagent groove 1,8 reagent bottles are divided into three row from the left surface of box body to right flank, wherein,
A row positioned at left surface include 3 reagent bottles, and the row positioned at right flank include 3 reagent bottles, the Yi Liebao positioned at centre
Containing 2 reagent bottles, trailing flank that the powdered reagent groove 1 is fixed in box body.
In the utility model, the side being connected with lid is designated as trailing flank, and the side relative with the trailing flank is front side
Door, the side on the front side door left side are left surface, and the side on the right of the front side door is right flank.
Preferably, the row positioned at left surface are followed successively by the examination of the anti-p16 albumen primary antibody reagent of instant from front to back
The reagent bottle 2 of agent bottle 4, the reagent bottle 3 of instant confining liquid and instant peroxidase inhibitors.
In the utility model, " instant " be directly used in the reagent of detection for that can be matched without dilution or adjustment.
Preferably, it is described positioned at right flank one row be followed successively by from front to back instant haematoxylin dyeing liquid reagent bottle 7,
The reagent bottle 6 of secondary antibody reagent and the reagent bottle 5 of increased response liquid of instant HRP marks.
In the utility model, " HRP " is horseradish peroxidase.
Preferably, the reagent bottle 8 and DAB dilutions for being followed successively by DAB concentrates from front to back positioned at a middle row
Reagent bottle 9.
In the utility model, " DAB " is diaminobenzidine.
Preferably, the reagent bottle 4 of the anti-p16 albumen primary antibody reagent of the instant, the reagent bottle 3 of instant confining liquid, i.e.
The secondary antibody marked with the reagent bottle 2 of type peroxidase inhibitors, the reagent bottle 7 of instant haematoxylin dyeing liquid, instant HRP
The volume of the reagent bottle 8 of the reagent bottle 6 of reagent, the reagent bottle 5 of increased response liquid and DAB concentrates is 1ml.
Preferably, the volume of reagent bottle 9 of the DAB dilutions is 5ml.
Preferably, the powdered reagent groove 1 is used to place PBS pulvis.
The portion of reagent can be prepared by the following method to obtain：
(1) the anti-p16 albumen primary antibody reagent of instant：2ug/ml.
(2) instant confining liquid：3%BSA.
(3) instant peroxidase inhibitors：3%H2O2。
(4) the secondary antibody reagent of instant HRP marks：0.5ug/ml.
In the utility model, the specifically used method of the kit is as follows：
1st, the sample process before immunohistochemical staining
(1) cell climbing sheet growth will be carried out in testing sample culture to slide, the cell density of growth reaches 80%-
Between 90%；
(2) the PBS pulvis in powdered reagent groove 1 is configured to 1L PBS cushioning liquid with distilled water, cell is washed with PBS
Slide 3 times, each 20s, 10min is fixed after drying with 95% ethanol room temperature；
(3) cell slide 3 times, each 20s is washed with PBS, is carried out carefully with 0.5%Triton X-100 after drying PBS
Penetrating processing (room temperature) 5min of after birth；
(4) wash cell slide 3 times, each 20s with PBS, dry PBS, the sample process before immunohistochemical staining is complete
Into,；
2nd, immunohistochemical staining
(1) the instant peroxidase inhibitors added on cell slide in appropriate reagent bottle 2, are placed in 37 DEG C
15min is incubated in constant water bath box；
(2) cell slide 3 times, each 20s is washed with PBS, dries PBS, appropriate examination is added on cell slide
Instant confining liquid in agent bottle 3, it is placed in 37 DEG C of constant water bath box and is incubated 20min；
(3) drying confining liquid, the one of the anti-p16 albumen of instant added on cell slide in appropriate reagent bottle 4
It is anti-, it is placed in 37 DEG C of constant water bath box and is incubated 15min；
(4) cell slide 3 times, each 20s is washed with PBS, dries PBS, appropriate examination is added on cell slide
Increased response liquid in agent bottle 5, it is placed in 37 DEG C of constant water bath box and is incubated 5min；
(5) cell slide 3 times, each 20s is washed with PBS, dries PBS, appropriate examination is added on cell slide
Instant HRP mark secondary antibodies in agent bottle 6, are placed in 37 DEG C of constant water bath box and are incubated 5min；
(6) cell slide 3 times, each 20s is washed with PBS, by the DAB concentrates and reagent bottle 9 in reagent bottle 8
DAB dilutions are according to 1:5 ratio is prepared, and the appropriate DAB nitrite ions prepared are added on cell slide and are carried out
Develop the color 3min；
(7) running water washes clean is used, the instant Soviet Union added after drying on cell slide in appropriate reagent bottle 7
Lignin dyeing liquor redyes 1min；
(8) running water washes clean is used, water-based gummy mounting after drying, micro- Microscopic observation result.
In the utility model, it is described it is " appropriate " be 50-200 μ l, those skilled in the art can be according to detection sample
Demand is adjusted, and does not do particular determination herein, and the number of reagent addition will not cause to testing result of the present utility model
Influence.
Relative to prior art, the utility model has the advantages that：
The utility model makes public for the first time a kind of quick detection kit for detecting people's p16 albumen, can qualitative analysis cell
Or people's p16 albumen in tissue sample, it is adapted to industrialized production；All related reagents in the utility model kit are all
The instant working solution prepared, reduce unnecessary troublesome operation step, compared to the immunohistochemical experiment of routine, during detection
Between shorten half, so as to reach quickly and easily detect p16 albumen effect.
Brief description of the drawings
Fig. 1 is the quick detection kit schematic diagram for detecting people's p16 albumen, wherein, reagent is respectively in figure：1-PBS powder
Agent, 2- instant peroxidase inhibitors, 3- instant confining liquids, the primary antibody of the anti-p16 albumen of 4- instants, 5- increased responses
Liquid, the secondary antibody of 6- instants HRP marks, 7- instant haematoxylin dyeing liquid, 8-DAB concentrates, 9-DAB dilutions；
Fig. 2 is to carry out kit immunohistochemical staining result with the positive Siha cervical cancer cells strains of HPV18.
Embodiment
The technical solution of the utility model is further illustrated below by embodiment.Those skilled in the art should
This understands that the embodiment is only to aid in understanding the utility model, is not construed as to concrete restriction of the present utility model.
Embodiment 1
As shown in figure 1, detection kit of the present utility model includes being provided with 8 reagent bottles and 1 powdered reagent in box body
Groove 1,8 reagent bottles are divided into three row from the left surface of box body to right flank, and the row positioned at left surface are from front to back
It is followed successively by reagent bottle 4, the reagent bottle 3 and instant peroxide of instant confining liquid of the anti-p16 albumen primary antibody reagent of instant
The reagent bottle 2 of enzyme inhibitor, the row positioned at right flank are followed successively by the reagent of instant haematoxylin dyeing liquid from front to back
The reagent bottle 6 of secondary antibody reagent and the reagent bottle 5 of increased response liquid of bottle 7, instant HRP marks, it is described positioned at a middle row
The reagent bottle 8 of DAB concentrates and the reagent bottle 9 of DAB dilutions are followed successively by from front to back, and the powdered reagent groove 1 is fixed on box
Internal trailing flank, for placing PBS pulvis.
Reagent bottle 4, reagent bottle 3, the instant peroxide of instant confining liquid of the anti-p16 albumen primary antibody reagent of instant
The examination for the secondary antibody reagent that the reagent bottle 2 of compound enzyme inhibitor, the reagent bottle 7 of instant haematoxylin dyeing liquid, instant HRP are marked
The volume of the reagent bottle 8 of agent bottle 6, the reagent bottle 5 of increased response liquid and DAB concentrates is 1ml.
The volume of reagent bottle 9 of the DAB dilutions is 5ml.
The portion of reagent can be prepared by the following method to obtain：
(1) the anti-p16 albumen primary antibody reagent of instant：2ug/ml.
(2) instant confining liquid：3%BSA.
(3) instant peroxidase inhibitors：3%H2O2。
(4) the secondary antibody reagent of instant HRP marks：0.5ug/ml.
In the utility model, the specifically used method of the kit is as follows：
1st, the sample process before immunohistochemical staining
(1) cell climbing sheet growth will be carried out in testing sample culture to slide, the cell density of growth reaches 80%-
Between 90%；
(2) the PBS pulvis in powdered reagent groove 1 is configured to 1L PBS cushioning liquid with distilled water, cell is washed with PBS
Slide 3 times, each 20s, 10min is fixed after drying with 4% paraformaldehyde room temperature；
(3) cell slide 3 times, each 20s is washed with PBS, is carried out carefully with 0.5%Triton X-100 after drying PBS
Penetrating processing (room temperature) 20min of after birth；
(4) wash cell slide 3 times, each 20s with PBS, dry PBS, the sample process before immunohistochemical staining is complete
Into,；
2nd, immunohistochemical staining
(1) the instant peroxidase inhibitors added on cell slide in 50 μ l reagent bottle 2, are placed in 37 DEG C
15min is incubated in constant water bath box；
(2) cell slide 3 times, each 20s is washed with PBS, dries PBS, 100 μ l examination is added on cell slide
Instant confining liquid in agent bottle 3, it is placed in 37 DEG C of constant water bath box and is incubated 20min；
(3) drying confining liquid, the one of the anti-p16 albumen of instant added on cell slide in 50 μ l reagent bottle 4
It is anti-, it is placed in 37 DEG C of constant water bath box and is incubated 15min；
(4) cell slide 3 times, each 20s is washed with PBS, dries PBS, 50 μ l examination is added on cell slide
Increased response liquid in agent bottle 5, it is placed in 37 DEG C of constant water bath box and is incubated 5min；
(5) cell slide 3 times, each 20s is washed with PBS, dries PBS, 50 μ l examination is added on cell slide
Instant HRP mark secondary antibodies in agent bottle 6, are placed in 37 DEG C of constant water bath box and are incubated 5min；
(6) cell slide 3 times, each 20s is washed with PBS, by the DAB concentrates and reagent bottle 9 in reagent bottle 8
DAB dilutions are according to 1:20 ratio is prepared, and the DAB nitrite ions prepared that 50 μ l are added on cell slide are carried out
Develop the color 3min；
(7) running water washes clean is used, the instant Soviet Union added after drying on cell slide in 50 μ l reagent bottle 7
Lignin dyeing liquor redyes 2min；
(8) running water washes clean is used, water-based gummy mounting after drying, micro- Microscopic observation result.
As a result as shown in Fig. 2 the cytoplasm and nucleus of Siha cells show obvious brown, Siha cells height is shown
Express P16 albumen.
Applicant states that the utility model illustrates people P16 protein determinations examination of the present utility model by above-described embodiment
Agent box, but the utility model is not limited to above-mentioned embodiment, that is, does not mean that the utility model has to rely on above-mentioned implementation
Mode could be implemented.Person of ordinary skill in the field is new to this practicality it will be clearly understood that to any improvement of the present utility model
The equivalence replacement of raw material selected by type and the addition of auxiliary element, the selection of concrete mode etc., all fall within guarantor of the present utility model
Protect within the scope of scope and disclosure.
Claims (7)
1. a kind of detection kit of people p16 albumen, including box body and the clamshell lid that is connected with box body, its feature exist
In with the groove and the reagent bottle on low groove for placing reagent bottle in the box body, the box is interior to be provided with 8 reagent bottles
With 1 powdered reagent groove (1), 8 reagent bottles are divided into three row from the left surface of box body to right flank, wherein, positioned at left side
One row in face include 3 reagent bottles, and the row positioned at right flank include 3 reagent bottles, and the row positioned at centre include 2 reagents
Bottle, the trailing flank that the powdered reagent groove (1) is fixed in box body.
2. detection kit according to claim 1, it is characterised in that it is described positioned at left surface one row from front to back according to
It is secondary for the reagent bottle (4) of the anti-p16 albumen primary antibody reagent of instant, the reagent bottle (3) of instant confining liquid and instant peroxidating
The reagent bottle (2) of thing enzyme inhibitor.
3. detection kit according to claim 2, it is characterised in that it is described positioned at right flank one row from front to back according to
The reagent bottle (6) for the secondary antibody reagent that secondary reagent bottle (7), instant HRP for instant haematoxylin dyeing liquid is marked and reaction increase
The reagent bottle (5) of strong liquid.
4. detection kit according to claim 3, it is characterised in that described to be arranged from front to back successively positioned at middle one
The reagent bottle (9) of reagent bottle (8) and DAB dilutions for DAB concentrates.
5. detection kit according to claim 4, it is characterised in that the anti-p16 albumen primary antibody reagent of instant
Reagent bottle (4), the reagent bottle (3) of instant confining liquid, the reagent bottle (2) of instant peroxidase inhibitors, instant Soviet Union
Reagent bottle (6), the reagent bottle of increased response liquid of the reagent bottle (7) of lignin dyeing liquor, the secondary antibody reagent of instant HRP marks
(5) and the volume of the reagent bottle (8) of DAB concentrates is 1ml.
6. detection kit according to claim 4, it is characterised in that the volume of the reagent bottle (9) of the DAB dilutions
For 5ml.
7. detection kit according to claim 1, it is characterised in that the powdered reagent groove (1) is used to place PBS powder
Agent.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201720323879.2U CN206975044U (en) | 2017-03-30 | 2017-03-30 | A kind of detection kit of people p16 albumen |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201720323879.2U CN206975044U (en) | 2017-03-30 | 2017-03-30 | A kind of detection kit of people p16 albumen |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108760450A (en) * | 2018-07-18 | 2018-11-06 | 杭州依美洛克医学科技有限公司 | Secondary antibody coloring system and colouring method for immunohistochemistry autostainer |
-
2017
- 2017-03-30 CN CN201720323879.2U patent/CN206975044U/en not_active Ceased
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108760450A (en) * | 2018-07-18 | 2018-11-06 | 杭州依美洛克医学科技有限公司 | Secondary antibody coloring system and colouring method for immunohistochemistry autostainer |
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| GR01 | Patent grant | ||
| IW01 | Full invalidation of patent right |
Decision date of declaring invalidation: 20210125 Decision number of declaring invalidation: 47823 Granted publication date: 20180206 |
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