CN110346552A - A kind of universal antigen retrieval buffer - Google Patents
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- 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
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- 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
- G01N1/44—Sample treatment involving radiation, e.g. heat
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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
- G01N33/531—Production of immunochemical test materials
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Abstract
The invention discloses a kind of universal antigen retrieval buffers, which is characterized in that it includes citraconic anhydride 0.5g/L；Nonionic surface active agent 5ml/L；Its production method are as follows: for preparing one liter of buffer: weighing citraconic anhydride 0.5g, take 1000ml beaker, by weighed 0.5g citraconic anhydride, be dissolved in the distilled water of 800ml, stir and evenly mix；The nonionic surface active agent of 5ml is added in solution after mixing, stirs and evenly mixs；PH value is adjusted to 7.4 with the NaOH solution that equivalent concentration is 1N；Then 1000ml graduated cylinder is taken, the solution that pH value is 7.4 is poured into graduated cylinder, is settled to 1000ml with distilled water.Universal antibody provided by the present invention repairs liquid, then can be applied to the antibody for carrying out antigen retrieval again using hot repair of the overwhelming majority, can greatly facilitate the accurate application of pathology and scientific research immunohistochemistry antigen retrieval method.
Description
Technical field
The present invention relates to large biological molecule field of immunodetection, are applied in Immunohistochemical detection technology, especially relate to
And a kind of universal antigen retrieval buffer.
Background technique
Clinical disease natural sciences or biomedical basic scientific research unit at present, mostly use formaldehyde for conventional firming agent for specimen.Formaldehyde
Although fixing the corrupt self-dissolving after tissue can be prevented in vitro, the original form of histocyte is kept, and preferably save tissue
The antigenicity of internal large biological molecule substance；However, the amino in the aldehyde radical and tissue protein of formaldehyde, which forms crosslinking, to be made
Most of antigenic determinant is shielded, causes antibody that cannot effectively combine with target antigen, to influence immuning tissue
The testing result of chemistry, this bridging effect are aggravated with fixed time incrementing.Tissue antigen determinant caused by fixed
Closing can be corrected with crosslinking by the method for antigen retrieval.Antigen retrieval method can substantially be divided into heating reparation and enzyme disappears
Change and repairs.
I. enzymic digestion antigen retrieval.
Immunohistochemistry antigen retrieval technology development early stage a kind of common method, general operation is using protease working solution
5~20min of histotomy is digested at 37 DEG C.Common protease is mainly trypsase, pepsin, Proteinase K etc..Pancreas egg
White enzyme and Proteinase K are chiefly used in intracellular antigen；Pepsin is then chiefly used in the reparation of cytoplasm antigen.
II. heating is repaired
1. according to the difference of the mode of heating, and immersion method, microwave heating method, hyperbaric heating method can be divided into.
2. repairing buffer: acid, neutral and alkaline buffer can be divided by repairing buffer according to the difference of pH value.At present
Common buffer has following several: citrate buffer solution (pH6.0), edta buffer liquid (pH8.0), Tris-Hcl buffer
(pH10.0), Tris-EDTA buffer (pH9.0) etc..Generally on the primary antibody specification that can be used as paraffin section immunohistochemistry,
It can indicate the antigen retrieval buffer of recommendation.To in antigen retrieval buffer now, different antibody need to use different resist
Original repairs buffer.There are many immunohistochemistry antibody type that clinical disease natural sciences use, and different suppliers is also many both at home and abroad, this
The counterpart application of sample difference antigen retrieval buffers and miscellaneous antibody significantly increases the workload of clinical disease natural sciences, and
If also will lead to the risk of detection failure using mistake antigen retrieval buffers.
Due to making clinical pathology immunohistochemistry detect work using different a variety of antigen retrieval buffers for different antibodies
Risk that is very inconvenient, and having sizable application error that detection is brought to fail.In existing a variety of antigen retrieval buffers, all can only
Applied to partial antibody.Universal antibody provided by the present invention repairs liquid, then can be applied to the overwhelming majority uses heat
The antibody for carrying out antigen retrieval is repaired, pathology can be greatly facilitated and the accurate of scientific research immunohistochemistry antigen retrieval method answers
With.
Summary of the invention
In order to overcome the drawbacks described above of the prior art, the present invention provides a kind of universal antigen retrieval buffer.Its feature
It is comprising citraconic anhydride 0.5g/L；Nonionic surface active agent 5ml/L；Its production method are as follows: to prepare one liter of buffer
For: citraconic anhydride 0.5g is weighed, 1000ml beaker is taken, by weighed 0.5g citraconic anhydride, is dissolved in the distilled water of 800ml, stirs
Mix mixing；The nonionic surface active agent of 5ml is added in solution after mixing, stirs and evenly mixs；It is 1N's with equivalent concentration
NaOH solution adjusts pH value to 7.4；Then 1000ml graduated cylinder is taken, the solution that pH value is 7.4 is poured into graduated cylinder, it is fixed with distilled water
Hold to 1000ml.
Preferably, the nonionic surface active agent is polysorbas20.
A kind of universal antigen retrieval buffer restorative procedure, which comprises the following steps:
Step 1: histotomy being taken out, carries out detection correspondence markings in the frosting of slice with pencil；Slice is put into
It is placed in Constant Temp. Oven in dyeing of plastics frame, is baked under conditions of 60 DEG C piece 1 hour.
Step 2: histotomy being sequentially placed into dewaxing liquid I, II, III and is impregnated respectively 5 minutes；Graded ethanol (concentration by
High to Low 100%~100%~95%~75%) respectively impregnates 2 minutes, pure water rinsing 30 seconds.
Step 3: histotomy is placed in 3% aqueous hydrogen peroxide solution (ready-to-use) and is impregnated 5 minutes, pure water rinsing
30 seconds.
Step 4: slice is put into the reparation box equipped with antigen retrieval buffer, then reparation box is put into water-bath,
Water-bath lid is covered, is begun to warm up；After water boiling, start timing, after twenty minutes, stops heating, taken out from water-bath
Reparation box, Temperature fall 30 minutes.Mark the region that blocks water: cleaning solution rinses 2 × 3 minutes.Get rid of the liquid on slide, then with inhaling
Water paper blots the liquid of tissue block edge, marks the region that blocks water along histotomy edge with immunohistochemistry pen.
Step 5: by the label on slice, primary antibody working solution is added dropwise and is allowed to be completely covered sample in right amount, in wet box at room temperature
It is incubated for 30 minutes.
Step 6: cleaning solution impregnates 2 × 5 minutes；Cleaning solution is got rid of, immune colour reagent is added dropwise, at room temperature (20~30 DEG C)
It is incubated for 20 minutes.
Step 7: cleaning solution impregnates 2 × 5 minutes；Cleaning solution is got rid of, DAB dyeing liquor is added dropwise and is allowed to that mark is completely covered in right amount
This, develops the color 5 minutes；Pure water rinsing color development stopping.
Step 8: getting rid of the extra liquid of slice surface, haematoxylin dyeing liquid is added dropwise and is allowed to that sample, room is completely covered in right amount
Temperature lower dyeing 3 minutes, 8~10 times were lifted with pickling solution and removes extra haematoxylin, pure water rinsing 30 seconds, PBS, which impregnates 3 minutes, to be returned
It is blue.
Step 9: slice successively respectively impregnates 2 minutes in graded ethanol (concentration from low to high 75%~95%~100%)；
Mounting is carried out using mountant, the slice after mounting is placed in sheet of drying in the air.
Step 10: the coloration result of microscopically observation slice.
Preferably, the dewaxing liquid is dimethylbenzene.
Preferably, the cleaning solution is phosphate buffer.
Preferably, the hydrochloride alcohol solution that the pickling solution is 1%.
Preferably, the immune colour reagent is that HRP marks mountain sheep anti mouse/two resistant polymers of rabbit.
Antigen retrieval buffer of the invention, by antigen retrieval effect and currently used concentration antigen retrieval buffer
Comparative experiments, it was demonstrated that antigen retrieval buffers of the invention have relatively good effect for the reparation of a variety of antigens.
The index of the antigen retrieval effect of test mainly select respectively karyon, endochylema, the after birth positive albumen (antigen)
Each 5 kinds, the selection details of corresponding antibody such as table 1: table 1: antibody selects details table
| Antibody | Clone number | Supplier | Dilution | Positive positioning |
| ki67 | MIB1 | Dako | 500 | Karyon |
| ER | SP1 | Dako | 100 | Karyon |
| PR | SP2 | Dako | 100 | Karyon |
| TTF-1 | 8G7G311 | Dako | 100 | Karyon |
| p53 | DO-7 | Dako | 300 | Karyon |
| CD4 | 1F6 | Leica | 20 | After birth |
| CD5 | 4C7 | Leica | 50 | After birth |
| CD10 | 56C6 | Leica | 50 | After birth |
| CD20 | L26 | Dako | 4000 | After birth |
| CK5/6 | D5/16B4 | Dako | 200 | Endochylema |
| CK20 | Ks20.8 | Leica | 40 | Endochylema |
| Melan-A | A103 | Dako | 20 | Endochylema |
| CK-pan | AE1/AE3 | Dako | 400 | Endochylema |
| SP-A | PE-10 | Dako | 100 | Endochylema |
Table 2: repairing effect contrast table
Coloration result is obtained by assessing positive tinctorial strength :-indicate negative；+ indicate weakly positive；++ indicate medium sun
Property；+++ indicate strong positive.Result data in comprehensive table, the hot antigen retrieval method of antigen retrieval solution, for these representatives
Property the antigen (each five kinds) for being positioned as karyon, after birth, endochylema, all there is good repairing effect, coloration result show, effect
Fruit is not weaker than any one of citrate buffer solution, edta buffer liquid, Tris-Hcl buffer, can be used as a general heat
The antigen retrieval solution of antigen retrieval method.
Universal antigen retrieval buffer proposed by the present invention passes through experimental verification, can effectively complete the overwhelming majority and exempt from
Epidemic disease groupization is operated using the antigen retrieval of antibody, and is configured and be easy, at low cost, is easy to use, is conducive to immunohistochemistry technique
Normalizing operation, offer convenience to user while reducing existing various antigen retrieval buffers and misuse and bring risk.
Below with reference to specific embodiment, the present invention will be described in detail.
Specific embodiment
Selection test organization is tonsillotome, (test object Ki67, Dako, 1:500)
1. configuring the citraconic anhydride solution of various concentration, specific concentration is as follows:
1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.001%, solvent is distilled water.
2. the citraconic anhydride solution under each concentration is adjusted to different pH value with hydrochloric acid and sodium hydroxide respectively, as follows: pH 2.0,
PH 7.4, pH 10 forms various concentration and the antigen retrieval buffer solution of difference PH.
Following steps are undergone, the process of antigen retrieval is completed.
Step 1: histotomy being taken out, carries out detection correspondence markings in the frosting of slice with pencil；Slice is put into
It is placed in Constant Temp. Oven in dyeing of plastics frame, is baked under conditions of 60 DEG C piece 1 hour.
Step 2: histotomy being sequentially placed into dewaxing liquid I, II, III and is impregnated respectively 5 minutes；Graded ethanol (concentration by
High to Low 100%~100%~95%~75%) respectively impregnates 2 minutes, pure water rinsing 30 seconds.
Step 3: histotomy is placed in 3% aqueous hydrogen peroxide solution (ready-to-use) and is impregnated 5 minutes, pure water rinsing
30 seconds.
Step 4: slice is put into the reparation box equipped with antigen retrieval buffer, then reparation box is put into water-bath,
Water-bath lid is covered, is begun to warm up；After water boiling, start timing, after twenty minutes, stops heating, taken out from water-bath
Reparation box, Temperature fall 30 minutes.Mark the region that blocks water: cleaning solution rinses 2 × 3 minutes.Get rid of the liquid on slide, then with inhaling
Water paper blots the liquid of tissue block edge, marks the region that blocks water along histotomy edge with immunohistochemistry pen.
Step 5: by the label on slice, primary antibody working solution is added dropwise and is allowed to be completely covered sample in right amount, in wet box at room temperature
It is incubated for 30 minutes.
Step 6: cleaning solution impregnates 2 × 5 minutes；Cleaning solution is got rid of, immune colour reagent is added dropwise, at room temperature (20~30 DEG C)
It is incubated for 20 minutes.
Step 7: cleaning solution impregnates 2 × 5 minutes；Cleaning solution is got rid of, DAB dyeing liquor is added dropwise and is allowed to that mark is completely covered in right amount
This, develops the color 5 minutes；Pure water rinsing color development stopping.
Step 8: getting rid of the extra liquid of slice surface, haematoxylin dyeing liquid is added dropwise and is allowed to that sample, room is completely covered in right amount
Temperature lower dyeing 3 minutes, 8~10 times were lifted with pickling solution and removes extra haematoxylin, pure water rinsing 30 seconds, PBS, which impregnates 3 minutes, to be returned
It is blue.
Step 9: slice successively respectively impregnates 2 minutes in graded ethanol (concentration from low to high 75%~95%~100%)；
Mounting is carried out using mountant, the slice after mounting is placed in sheet of drying in the air.
Slice after treatment is observed, is obtained following as a result, such as table 3:
Table 3:
Coloration result is obtained by assessing positive tinctorial strength:
Indicate negative；+ indicate weakly positive；++ indicate moderate positive；+++ indicate strong positive
The result of various concentration and difference PH comparison in comprehensive table show that the concentration of preferred citraconic anhydride solution is
0.05% i.e. 0.5g/L；Preferred pH value is 7.4.
Although combining preferred embodiment above, the present invention is described, ordinary skill in the art
Personnel are it should be appreciated that above-mentioned example is intended merely to explanation, and cannot function as limitation of the present invention.Therefore, it can weigh
It modifies in the spirit of sharp claim to the present invention and modification, these modifications and variations will all fall in of the invention
Within the scope of claims are required.
Claims (7)
1. a kind of universal antigen retrieval buffer, which is characterized in that it includes citraconic anhydride 0.5g/L；Non-ionic surfactant
Agent 5ml/L；Its production method are as follows: for preparing one liter of buffer: weighing citraconic anhydride 0.5g, take 1000ml beaker, will weigh
0.5g citraconic anhydride, be dissolved in the distilled water of 800ml, stir and evenly mix；The non-ionic of 5ml is added in solution after mixing
Surfactant stirs and evenly mixs；PH value is adjusted to 7.4 with the NaOH solution that equivalent concentration is 1N；Then 1000ml graduated cylinder is taken, it will
The solution that pH value is 7.4 pours into graduated cylinder, is settled to 1000ml with distilled water.
2. universal antigen retrieval buffer according to claim 1, it is characterised in that: the non-ionic surfactant
Agent is polysorbas20.
3. a kind of universal antigen retrieval buffer restorative procedure described in claim 1, which is characterized in that including following step
It is rapid:
Step 1: histotomy being taken out, carries out detection correspondence markings in the frosting of slice with pencil；Slice is put into plastics
It is placed in Constant Temp. Oven in staining rack, is baked under conditions of 60 DEG C piece 1 hour.
Step 2: histotomy being sequentially placed into dewaxing liquid I, II, III and is impregnated respectively 5 minutes；Graded ethanol (concentration by height to
Low 100%~100%~95%~75%) respectively impregnates 2 minutes, pure water rinsing 30 seconds.
Step 3: histotomy is placed in 3% aqueous hydrogen peroxide solution (ready-to-use) and is impregnated 5 minutes, pure water rinsing 30
Second.
Step 4: slice being put into the reparation box equipped with antigen retrieval buffer, then reparation box is put into water-bath, is covered
Water-bath lid, begins to warm up；After water boiling, start timing, after twenty minutes, stops heating, take out and repair from water-bath
Box, Temperature fall 30 minutes.Mark the region that blocks water: cleaning solution rinses 2 × 3 minutes.The liquid on slide is got rid of, then uses blotting paper
The liquid for blotting tissue block edge marks the region that blocks water along histotomy edge with immunohistochemistry pen.
Step 5: by the label on slice, primary antibody working solution is added dropwise and is allowed to that sample is completely covered in right amount, is incubated at room temperature in wet box
30 minutes.
Step 6: cleaning solution impregnates 2 × 5 minutes；Cleaning solution is got rid of, immune colour reagent is added dropwise, at room temperature (20~30 DEG C) incubations
20 minutes.
Step 7: cleaning solution impregnates 2 × 5 minutes；Cleaning solution is got rid of, DAB dyeing liquor is added dropwise and is allowed to that sample is completely covered in right amount, shows
Color 5 minutes；Pure water rinsing color development stopping.
Step 8: getting rid of the extra liquid of slice surface, haematoxylin dyeing liquid is added dropwise and is allowed to that sample is completely covered in right amount, at room temperature
Dyeing 3 minutes lifts 8~10 times with pickling solution and removes extra haematoxylin, and pure water rinsing 30 seconds, PBS, which impregnates 3 minutes, returned indigo plant.
Step 9: slice successively respectively impregnates 2 minutes in graded ethanol (concentration from low to high 75%~95%~100%)；It uses
Mountant carries out mounting, and the slice after mounting is placed in sheet of drying in the air.
4. universal antigen retrieval buffer restorative procedure according to claim 3, which is characterized in that the dewaxing liquid is
Dimethylbenzene.
5. universal antigen retrieval buffer restorative procedure according to claim 3, which is characterized in that the cleaning solution is
Phosphate buffer.
6. universal antigen retrieval buffer restorative procedure according to claim 3, which is characterized in that the pickling solution is
1% hydrochloride alcohol solution.
7. universal antigen retrieval buffer restorative procedure according to claim 3, which is characterized in that the immune colour developing
Reagent is that HRP marks mountain sheep anti mouse/two resistant polymers of rabbit.
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