CN108535076A - A kind of confining liquid and preparation method thereof applied to immunocytochemistry P16 protein stainings - Google Patents
A kind of confining liquid and preparation method thereof applied to immunocytochemistry P16 protein stainings Download PDFInfo
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- CN108535076A CN108535076A CN201810224854.6A CN201810224854A CN108535076A CN 108535076 A CN108535076 A CN 108535076A CN 201810224854 A CN201810224854 A CN 201810224854A CN 108535076 A CN108535076 A CN 108535076A
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- 239000007788 liquid Substances 0.000 title claims abstract description 75
- 238000010186 staining Methods 0.000 title claims abstract description 22
- 238000003365 immunocytochemistry Methods 0.000 title claims abstract description 21
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims description 7
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims abstract description 34
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 34
- 235000020183 skimmed milk Nutrition 0.000 claims abstract description 29
- 239000012153 distilled water Substances 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 17
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000001103 potassium chloride Substances 0.000 claims abstract description 17
- 235000011164 potassium chloride Nutrition 0.000 claims abstract description 17
- 239000011780 sodium chloride Substances 0.000 claims abstract description 17
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 16
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 16
- 108010088751 Albumins Proteins 0.000 claims description 6
- 102000009027 Albumins Human genes 0.000 claims description 6
- 239000012137 tryptone Substances 0.000 claims description 5
- RDEIXVOBVLKYNT-HDZPSJEVSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-[(1r)-1-aminoethyl]oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;(2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(aminomethyl)oxan-2 Chemical compound OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@@H](CN)O2)N)[C@@H](N)C[C@H]1N.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@H](O2)[C@@H](C)N)N)[C@@H](N)C[C@H]1N.O1[C@H]([C@@H](C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N RDEIXVOBVLKYNT-HDZPSJEVSA-N 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 27
- 238000007789 sealing Methods 0.000 abstract description 22
- 238000004043 dyeing Methods 0.000 abstract description 11
- 238000003364 immunohistochemistry Methods 0.000 abstract description 8
- 235000018102 proteins Nutrition 0.000 abstract description 8
- 241001465754 Metazoa Species 0.000 abstract description 7
- 210000002966 serum Anatomy 0.000 abstract description 7
- 239000005018 casein Substances 0.000 abstract description 4
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 abstract description 4
- 235000021240 caseins Nutrition 0.000 abstract description 4
- 208000015181 infectious disease Diseases 0.000 abstract description 3
- 241000700605 Viruses Species 0.000 abstract description 2
- 239000013641 positive control Substances 0.000 description 11
- 238000012360 testing method Methods 0.000 description 6
- 208000019065 cervical carcinoma Diseases 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 235000013861 fat-free Nutrition 0.000 description 4
- 235000013336 milk Nutrition 0.000 description 4
- 239000008267 milk Substances 0.000 description 4
- 210000004080 milk Anatomy 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 230000009471 action Effects 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- 208000034254 Squamous cell carcinoma of the cervix uteri Diseases 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 201000006612 cervical squamous cell carcinoma Diseases 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 210000003097 mucus Anatomy 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 208000031295 Animal disease Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 210000004996 female reproductive system Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
Abstract
The present invention provides a kind of confining liquid applied to immunocytochemistry P16 protein stainings, and the confining liquid includes following component by weight：40 50 parts of skimmed milk power；7 10 parts of sodium chloride；0.1 0.3 parts of potassium chloride；1.0 2.0 parts of disodium hydrogen phosphate；0.2 0.4 parts of potassium dihydrogen phosphate；1000 parts of distilled water；The confining liquid off-period is 15 40min, and closure temperature is 32~35 DEG C.Inventive closure liquid can close non-specific background's dyeing, and will not cause signal weaker of developing caused by excessively closing.Compared to immunohistochemistry confining liquids such as more common BSA, casein, animal non-immune serums, confining liquid sealing effect of the invention is more preferable, and cost is relatively low, while also without the risk of infection animal virus.
Description
Technical field
The invention belongs to clinical medicine pathology technique fields, and in particular to one kind being applied to immunocytochemistry P16 albumen
Confining liquid of dyeing and preparation method thereof.
Background technology
Cervical carcinoma is one of most common female reproductive system malignant tumour of gynaecology, and incidence is only second to breast cancer, occupies
Female malignant second seriously threatens the health and quality of life of women.China is the country occurred frequently of cervical carcinoma, hair
Raw rate and the death rate account for about global 1/3.Cervical carcinoma is also that the currently the only cause of disease is clear, by effectively early diagnosing and controlling
Treat the cancer that can be cured completely.
Currently, cervical carcinoma screening generally uses liquid based cytology, and liquid based cytology specificity is high by about 90%, but sensitivity is not
Foot, about 50%, cause about half patient's missing inspection.To make up liquid based cytology deficiency, clinical application HPV genetic tests are as auxiliary
Technology is helped, HPV genetic test sensitivity is higher by about 90%, but specificity easily causes over-treatment and medical resource less than 10%
Waste.The U.S. in 2012, World Health Organization in 2014 confirm that P16 is the unique biomarker of cervical carcinoma.Cervical exfoliated cell is exempted from
Epidemic disease cytochemistry P16 protein stainings compare liquid based cytology and HPV genetic tests, with 90% or more sensitivity, and specifically
Property is higher by about 85%, and clinical meaning is clear.
In immunohistochemical reaction, antibody can identify antigenic determinant, then specifically bind therewith.Confining liquid
Action principle is mainly non-specifically combined with the antigenic determinant on surface by albumen, causes specific antibody cannot be with
These nonspecific antigenic determinants combine, and dyeing background will reduce.And target antigen determinant is combined very with antibody
Specifically, binding ability is very strong, can still be combined after closing.But if confining liquid off-period is too long or excessive concentration,
The specific binding that can influence antibody leads to signal weaker of finally developing.The common confining liquid of immunohistochemistry has serum, junket
Albumen etc., if patent CN104990781A uses lowlenthal serum as confining liquid, 37 DEG C are incubated 10 minutes；Patent
CN203350254U uses instant BSA confining liquids, is incubated at room temperature 1 hour or 4 DEG C overnight；Patent CN102183651A is used
Animal blood serum, albumin etc. are used as confining liquid, and 37 DEG C are incubated 40 minutes.
In image formation, the action principle of confining liquid is identical with immunohistochemistry, however cell sheet and
The difference of tissue makes the common confining liquid of immunohistochemistry and off-period be not suitable for being directly used in immunocytochemistry,
The immunocytochemical stain kit as disclosed in patent CN102565393A prepares confining liquid using BSA, fetal calf serum, text
In do not refer to the sealing effect of confining liquid, and in fact the confining liquid easily causes to close the incomplete non-specific back of the body
Scape dyes, or the development signal weaker that closing is excessive.Patent CN105241726A discloses confining liquid group and is divided into skimmed milk power
45-55g, potassium dihydrogen phosphate 0.25-0.29g, disodium hydrogen phosphate 1.32-1.52g, sodium chloride 7-9g, potassium chloride 0.15-0.25g,
Distilled water 1L, but in file the sealing effect of confining liquid is not more referred to there is no the off-period of open confining liquid and temperature.
Currently, P16 protein immunocytochemistries dyeing in the country's, in the initial stage of applying, a kind of suitable confining liquid of exploration pushes away the technology
Wide application plays very important effect.
Invention content
The object of the present invention is to provide a kind of confining liquid applied to immunocytochemistry P16 protein stainings, the closings
Liquid energy completely encloses non-specific background's dyeing, and will not cause signal weaker of developing caused by excessively closing, and ensures best
Sealing effect.
Another object of the present invention is to provide the systems of the confining liquid applied to immunocytochemistry P16 protein stainings
Preparation Method.
The above-mentioned purpose of the present invention is achieved by the following technical programs：
A kind of confining liquid applied to immunocytochemistry P16 protein stainings, the confining liquid include it is following by weight
Count component：
40-50 parts of skimmed milk power；
7-10 parts of sodium chloride；
0.1-0.3 parts of potassium chloride；
1.0-2.0 parts of disodium hydrogen phosphate；
0.2-0.4 parts of potassium dihydrogen phosphate；
1000 parts of distilled water；
The confining liquid off-period is 15-40min, and closure temperature is 32~35 DEG C.
Inventor is constantly the study found that the content of skimmed milk power is in 40-50g in the formula of confining liquid, sealing effect
It is optimal；When nonfat dry milk concentration is less than 40g, enclosed is decreased obviously；When nonfat dry milk concentration is higher than 50g, although enclosed is very
Height, but there is positive decrease phenomenon.And the off-period of confining liquid sealing effect in 15-40min is good, can close non-spy
Anisotropic background stainings, and signal weaker of developing caused by excessively closing will not be caused.Compared to more common BSA, casein, animal
The immunohistochemistry confining liquid such as non-immune serum, confining liquid sealing effect of the invention is more preferable, at low cost, and without infection animal disease
The risk of poison.
Preferably, the confining liquid includes following component by weight：
45-50 parts of skimmed milk power；
7-8 parts of sodium chloride；
0.15-0.25 parts of potassium chloride；
1.32-1.52 parts of disodium hydrogen phosphate；
0.25-0.29 parts of potassium dihydrogen phosphate；
1000 parts of distilled water.
Preferably, the confining liquid off-period is 25-40min, and closure temperature is 32~35 DEG C.Off-period is 25-
When 40min, the sealing effect of confining liquid is more preferably.
Preferably, the confining liquid further includes gentamicin sulphate 0.01-0.02 parts by weight, polysorbas20 0.5-1
Part.
Preferably, the confining liquid further includes albumin 15-20 parts by weight.
Preferably, the confining liquid further includes tryptone 10-12 parts by weight.
The preparation method of confining liquid of the present invention applied to immunocytochemistry P16 protein stainings, including walk as follows
Suddenly：
S1. sodium chloride, potassium chloride, disodium hydrogen phosphate, the potassium dihydrogen phosphate for weighing formula ratio, are dissolved in distilled water successively；
S2. skimmed milk power is added, skimmed milk power is added three times, and is stirred while adding and is made it dissolve uniformly, overnight；
S3. it filters, is sealed.
Preferably, S3 is filtered using Medium speed filter paper, and gained filtrate is sealed at 2-8 DEG C.
Application of the confining liquid in immunocytochemistry P16 dyeing.
Beneficial effects of the present invention：
1. the immunohistochemistry confining liquids such as the BSA that compares, casein, animal non-immune serum, confining liquid of the invention closing
Effect is more preferable, and cost is relatively low；
2. the confining liquids such as the BSA that compares, animal non-immune serum, without the risk of infection animal virus.
Description of the drawings
Fig. 1 is severe non-specific background's colored graph in 1 coloration result of embodiment；
Fig. 2 is moderate non-specific background's colored graph in 1 coloration result of embodiment；
Fig. 3 is 1 coloration result mild or moderate non-specific background's colored graph of embodiment；
Fig. 4 is in 1 coloration result of embodiment without non-specific background stainings figure；
Fig. 5 is the positive colour developing figure of positive control；
Fig. 6 is that the positive of positive control weakens and show negative figure.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with Figure of description and reality
Apply example, the present invention is described in more detail, but the present invention claims protection domain be not limited to embodiment.
The reagent specification that following embodiments use for：Sodium chloride (AR), potassium chloride (AR), disodium hydrogen phosphate (AR), phosphoric acid
Potassium dihydrogen (AR), distilled water (distilled water), gentamicin sulphate (USP grades), tryptone (BR grades), albumin (derive from chicken
Albumen, biotechnology grade).
Embodiment 1:
Sodium chloride 7g, potassium chloride 0.15g, disodium hydrogen phosphate 1.32g, potassium dihydrogen phosphate are weighed with electronic analytical balance
0.25g is dissolved in the distilled water of 1L successively, after dissolving completely, skimmed milk power 45g is added, skimmed milk power is added three times, Bian Jia
Enter side to stir to dissolve uniformly, after ambient temperature overnight, be filtered with Medium speed filter paper, 2-8 DEG C is sealed for use.
Embodiment 2:
Sodium chloride 8g, potassium chloride 0.25g, disodium hydrogen phosphate 1.52g, potassium dihydrogen phosphate are weighed with electronic analytical balance
0.29g is dissolved in the distilled water of 1L successively, after dissolving completely, skimmed milk power 50g is added, skimmed milk power is added three times, Bian Jia
Enter side to stir to dissolve uniformly, after ambient temperature overnight, be filtered with Medium speed filter paper, 2-8 DEG C is sealed for use.
Embodiment 3:
Sodium chloride 7g, potassium chloride 0.1g, disodium hydrogen phosphate 1.0g, potassium dihydrogen phosphate 0.20g are weighed with electronic analytical balance,
It is dissolved in the distilled water of 1L successively, after dissolving completely, skimmed milk power 40g is added, skimmed milk power is added three times, and is stirred when being added
It mixes and makes it dissolve uniformly, be then added sour gentamicin 0.01g, polysorbas20 0.5ml, after ambient temperature overnight, with Medium speed filter paper mistake
Filter, 2-8 DEG C is sealed for use.
Embodiment 4:
Sodium chloride 10g, potassium chloride 0.3g, disodium hydrogen phosphate 2.0g, potassium dihydrogen phosphate 0.4g are weighed with electronic analytical balance,
It is dissolved in the distilled water of 1L successively, after dissolving completely, skimmed milk power 50g is added, skimmed milk power is added three times, and is stirred when being added
It mixing and makes it dissolve uniformly, be then added sour gentamicin 0.02g, polysorbas20 1.0g after ambient temperature overnight, is filtered with Medium speed filter paper,
2-8 DEG C is sealed for use.
Embodiment 5:
Sodium chloride 7g, potassium chloride 0.15g, disodium hydrogen phosphate 1.32g, potassium dihydrogen phosphate are weighed with electronic analytical balance
0.25g, albumin 15g are dissolved in the distilled water of 1L successively, and after dissolving completely, skimmed milk power 45g, skimmed milk power point 3 times is added
It is added, stirs while adding and make it dissolve uniformly, after ambient temperature overnight, filtered with Medium speed filter paper, 2-8 DEG C is sealed for use.
Embodiment 6：
Sodium chloride 7g, potassium chloride 0.15g, disodium hydrogen phosphate 1.32g, potassium dihydrogen phosphate are weighed with electronic analytical balance
0.25g, albumin 20g are dissolved in the distilled water of 1L successively, and after dissolving completely, skimmed milk power 45g, skimmed milk power point 3 times is added
It is added, stirs while adding and make it dissolve uniformly, after ambient temperature overnight, filtered with Medium speed filter paper, 2-8 DEG C is sealed for use.
Embodiment 7：
Sodium chloride 7g, potassium chloride 0.15g, disodium hydrogen phosphate 1.32g, potassium dihydrogen phosphate are weighed with electronic analytical balance
0.25g, tryptone 10g are dissolved in the distilled water of 1L successively, and after dissolving completely, skimmed milk power 45g, skimmed milk power point 3 is added
Secondary addition is stirred while adding and is made it dissolve uniformly, after ambient temperature overnight, filtered with Medium speed filter paper, and 2-8 DEG C is sealed for use.
Embodiment 8：
Sodium chloride 7g, potassium chloride 0.15g, disodium hydrogen phosphate 1.32g, potassium dihydrogen phosphate are weighed with electronic analytical balance
0.25g, tryptone 12g are dissolved in the distilled water of 1L successively, and after dissolving completely, skimmed milk power 45g, skimmed milk power point 3 is added
Secondary addition is stirred while adding and is made it dissolve uniformly, after ambient temperature overnight, filtered with Medium speed filter paper, and 2-8 DEG C is sealed for use.
Effect example
One, confining liquid compliance test result is tested
1. experimental method and parameter：
After the completion of confining liquid configuration, immunocytochemistry is carried out to cervical exfoliated cell according to this field standard test flow
P16 is dyed.
(1) control group 1 is that immunohistochemistry often uses confining liquid 5%BSA solution；
(2) control group 2 is the highly purified casein confining liquid of immunohistochemistry of market purchasing；
(3) off-period selects 15min, 25min, 40min, 60min and 120min；Closure temperature is 32~35 DEG C.
(4) sample size be 496 samples, 124 samples of each time point, wherein have 62 samples contain more mucus and
Leucocyte.
(5) positive control uses cervical squamous cell carcinoma cell SiHa cells, and every group is respectively provided with 3 positive controls.
2. experimental result is explained as follows：
+++ there is strong non-specific background's dyeing, it can not judging result (shown in Fig. 1)；
++ there is moderate non-specific background dyeing, have larger impact to result judgement (shown in Fig. 2)；
+ there is slight unspecific staining, there is minimal effect to result judgement (shown in Fig. 3)；
Without non-specific background stainings (shown in Fig. 4)；
The positive control positive develops the color (shown in Fig. 5)；
The positive control positive weakens and display is negative (shown in Fig. 6).
Enclosed：Coloration result is+and result be-sum account for the ratios of 124 samples.
3. experimental result statistical analysis：
Table 1：Sealing effect when off-period is 15min
Table 2：Sealing effect when off-period is 25min
Table 3：Sealing effect when off-period is 40min
Table 4：Sealing effect when off-period is 60min
Table 5：Sealing effect when off-period is 120min
Table 6:More mucus and leucocyte sample closing result (n=248)
It can be seen that from table 1-6：
(1) when confining liquid off-period prepared by embodiment 1-8 is 15-40min, sealing effect is good；Wherein off-period
For 25-40min when sealing effect it is optimal；
(2) it can be obtained from table 4, table 5, when off-period is 60,120min, embodiment 1-8 when especially 120min
Confining liquid enclosed is up to 100%, and control group 1-2 encloseds are also increased to 90% or more, but embodiment 1-8 the positive occurs and subtracts
Weak, there is feminine gender in control group positive control, and positive signal obviously weakens, and indicates that closing is excessive.
(3) embodiment 1-8 confining liquids have good sealing effect, can meet Clinical practice requirement, sealing effect is apparent
Better than control group 1-2.
Two, influence experiment of the protein content to sealing effect
According to the action principle of confining liquid, suitable albumen concentration can guarantee best sealing effect, too low or excessively high
Concentration may cause sealing effect to decline or excessively close, signal weaker of developing.
1. test method：
(1) confining liquid is prepared according to the configuration method of embodiment 1, divides A, B, C, D, E, F totally 6 groups, wherein skimmed milk power
Content is respectively 30g, 35g, 40g, 45g, 50g, 55g.
(2) immunocytochemistry P16 dyeing is carried out to cervical exfoliated cell according to standard test flow.
(3) sample size is 124 samples, and each sample film-making 6 opens, and carries out parallel check experiment, use respectively A, B, C, D,
E, F confining liquids are closed, and other experimental conditions are consistent with parameter.
(4) according to experiment one, select off-period for 40min.
(5) positive control uses cervical squamous cell carcinoma cell SiHa cells, and every group is respectively provided with 3 positive controls.
2. experimental result is explained as follows：
+++ there is strong non-specific background's dyeing, it can not judging result；
++ there is moderate non-specific background dyeing, has larger impact to result judgement；
+ there is slight unspecific staining, there is minimal effect to result judgement；
Without non-specific background stainings；
The positive control positive develops the color；
The positive control positive weakens and display is negative.
3. experimental result statistical analysis：
Table 7：A, B, C, D, E, F confining liquid sealing effect count
It can be seen that according to table 7, when the content of skimmed milk power is in 40-50g, sealing effect is optimal；Nonfat dry milk concentration is low
When 40g, enclosed is decreased obviously；When nonfat dry milk concentration is higher than 50g, although enclosed is very high, there is positive decrease
Phenomenon.
According to the disclosure and teachings of the above specification, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula is changed and is changed.Therefore, the invention is not limited in specific implementation modes disclosed and described above, to the one of invention
A little modifications and changes should also be as falling into the scope of the claims of the present invention.In addition, although being used in this specification
Some specific terms, these terms are merely for convenience of description, does not limit the present invention in any way.
Claims (8)
1. a kind of confining liquid applied to immunocytochemistry P16 protein stainings, which is characterized in that the confining liquid includes as follows
Component by weight：
40-50 parts of skimmed milk power；
7-10 parts of sodium chloride；
0.1-0.3 parts of potassium chloride；
1.0-2.0 parts of disodium hydrogen phosphate；
0.2-0.4 parts of potassium dihydrogen phosphate；
1000 parts of distilled water；
The confining liquid off-period is 15-40min, and closure temperature is 32~35 DEG C.
2. being applied to the confining liquid of immunocytochemistry P16 protein stainings according to claim 1, which is characterized in that described
Confining liquid includes following component by weight：
45-50 parts of skimmed milk power；
7-8 parts of sodium chloride；
0.15-0.25 parts of potassium chloride；
1.32-1.52 parts of disodium hydrogen phosphate；
0.25-0.29 parts of potassium dihydrogen phosphate；
1000 parts of distilled water.
3. the confining liquid according to claim 1 or claim 2 applied to immunocytochemistry P16 protein stainings, which is characterized in that institute
It is 25-40min to state confining liquid off-period, and closure temperature is 32~35 DEG C.
4. the confining liquid according to claim 1 or claim 2 applied to immunocytochemistry P16 protein stainings, which is characterized in that institute
It further includes gentamicin sulphate 0.01-0.02 parts by weight, 0.5-1 parts of polysorbas20 to state confining liquid.
5. the confining liquid according to claim 1 or claim 2 applied to immunocytochemistry P16 protein stainings, which is characterized in that institute
It further includes albumin 15-20 parts by weight to state confining liquid.
6. the confining liquid according to claim 1 or claim 2 applied to immunocytochemistry P16 protein stainings, which is characterized in that institute
It further includes tryptone 10-12 parts by weight to state confining liquid.
7. the preparation method described in claim 1 applied to the confining liquid of immunocytochemistry P16 protein stainings, feature exist
In including the following steps：
S1. sodium chloride, potassium chloride, disodium hydrogen phosphate, the potassium dihydrogen phosphate for weighing formula ratio, are dissolved in distilled water successively；
S2. skimmed milk power is added, skimmed milk power is added three times, and is stirred while adding and is made it dissolve uniformly, overnight；
S3. it filters, is sealed.
8. being applied to the preparation method of the confining liquid of immunocytochemistry P16 protein stainings, feature according to claim 7
It is, S3 is filtered using Medium speed filter paper, and gained filtrate is sealed at 2-8 DEG C.
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