CN102435728A - Preparation method for positive control substance for inspection and control of quality in immunohistochemical process - Google Patents
Preparation method for positive control substance for inspection and control of quality in immunohistochemical process Download PDFInfo
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- CN102435728A CN102435728A CN2011102637311A CN201110263731A CN102435728A CN 102435728 A CN102435728 A CN 102435728A CN 2011102637311 A CN2011102637311 A CN 2011102637311A CN 201110263731 A CN201110263731 A CN 201110263731A CN 102435728 A CN102435728 A CN 102435728A
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Abstract
The invention provides a preparation method for a positive control substance for the inspection and control of quality in the immunohistochemical process. The method includes the following steps polypeptide or protein with different concentrations which can carry out specific reaction with an antibody are adsorbed on a slide in advance, or polypeptide or protein with different concentrations are placed on the slide in advance, the polypeptide or protein and a pathologic tissue section undergo a conventional immunohistochemical step at the same time, and the coloration result of the polypeptide or protein is used as positive control for the immunohistochemical process. The invention adopts the method of arranging the positive control protein or polypeptide on the slide to realize the positive control and a quality control standard, and the method is an important supplement to the existing quality assurance program, and is a new method for the quality control of immunohistochemical assays.
Description
Technical field
The invention belongs to field of pathology, particularly, the present invention relates to a kind of preparation method who is used for the positive object of reference of SABC procedure quality prosecution.
Background technology
Immunohistochemistry plays a part to become more and more important in biomedical research and diagnosed SARS case.Particularly from the nineties in last century, along with the appearance of high temperature antigen retrieval technology, the application in immunohistochemistry orthopaedic surgical operations pathology and other morphology fields more and more widely.In decades in the past,, become the indispensable instrument of medical diagnosis on disease in the pathology based on the immunohistochemistry technique of antigen retrieval, paraffin organization microtomy, antibody recognition.To the diagnosis of cancer, immunohistochemistry technique has become necessary means especially, is to diagnose out various various cancers main method at present clinically.
Quality control is the indispensable program of the quality of product of check or service, in the interpretation of result of SABC, most critical be how to confirm positive control.Positive control is one of most important test stone of whole process, realize through producing a kind of measurable signal, and sort signal receives the nearly all ingredient of SABC process, and the influence of technology.The content that the present invention relates to is the important supplement to existing quality assurance program, is that a kind of SABC is tested carried out new Method of Quality.
Summary of the invention
The object of the present invention is to provide a kind of preparation method that the SABC process is carried out the positive object of reference of quality that is used for.
Along with immunohistochemistry technique in clinical diagnosis and tumour Study on Transformation, the especially widespread use in the research and development of current cancer targeted therapy method, the medical personnel is more and more stronger to the requirement of IHC.Tumor marker and as the treatment indicator object of reference become two leading indicators that the IHC standardization relates to.Wherein, The selection of object of reference needs to create the corresponding standard object of reference to each tumor marker; Solve because the changeable IHC coloration result that inconsistent formalin fixed, repairing condition, immune response cause unitized, simultaneously as the quality standard of assessment sample preparations.Among the present invention, adopt polypeptide slide point sample matrix model standard as a reference, carry out the research of SABC quality control standard.
Can with the polypeptide of an anti-variable concentrations that specific immune response takes place in advance covalency be adsorbed on the slide, or be placed in advance on the slide through the polypeptide paraffin section of the variable concentrations of embedding, be used for follow-up placement patient's tissue specimen.Be adsorbed on the polypeptide and the histopathologic slide of slide, steps such as the elimination of the roasting sheet of experience, dewaxing, antigen retrieval, endogenous peroxydase simultaneously, sealing, an anti-reaction, two anti-reactions, colour developing.
Specific marker or have or do not have is the principal character that present tumour spectrum is identified.Rely on the appearance of immunohistochemical staining result's personalized treatment, increased the demand of positive control.The setting of positive control and negative control thing makes the influence that detects inconsistency minimize.On every slide, contrast, can tackle the risk that false wrong (like the reagent configuration of mistake, reagent actual effect, misoperation etc.) take place, also can effectively judge owing to the too high false positive results that causes of reagent sensitivity.
To positive object of reference (polypeptide) with a kind of disease target position, set up repeatably SABC polypeptide contrast of standardization, need differentiate the target position of the selected antibodies of the commerce of having used clinically.The polypeptide epitope of selecting has structure and the characteristic the same with the polypeptide of native antigen (epitope), and has antibody with antibodies and combine the same effect with native antigen.The polypeptide that uses is sayed from structure, must be the epi-position of native antigen, and differentiate that epitope has two universal methods: (1) is assessed the polypeptide of forming based on the native protein sequence; (2) polypeptide that the peptide library screening from combination is at random obtained is assessed.
The modes of emplacement of polypeptide on slide seen Fig. 1 or shown in Figure 2.Can with one anti-take place that immunoreactive polypeptide is printed or manual point sample on slide, the polypeptide of a concentration on the slide through on roundlet spot of formation after the appearance.Polypeptide among the figure can be that the set of the various variable concentrations of phase homopolypeptide is formed, and also can be the combination of a few peptide species different modes.
Technical scheme of the present invention comprises following process steps:
Can with the polypeptide or the albumen of antibody generation specific reaction; Variable concentrations is set to be adsorbed on the slide in advance; Or the polypeptide or the albumen paraffin section that will pass through the variable concentrations of embedding are placed on the slide in advance; Experience routine immunization group step simultaneously with histopathologic slide, the coloration result of polypeptide or albumen is as the positive object of reference of SABC process.
Described polypeptide or albumen are the epi-position of tissue antigen in the histopathologic slide.
Described polypeptide or albumen are adsorbed on the slide in advance, are the modes with covalency or physisorption, are adsorbed on the slide that SABC uses.
Described coloration result, the spot colors of polypeptide or albumen show gradually from concentration from low to high to be deepened; Through microscope histopathologic slide is observed again, judged according to the contrast of the spot colors of target position color and polypeptide or albumen in the histopathologic slide.
Quality Control Technology is very important in the Clinical detection application facet, for every kind of analysis that detects thing, needs a kind of tester of measuring at least.The control value of tester departs from the result of reservation, means that measuring the possibility of result mistake occurred, need reappraise to testing result.In immunohistochemical experiment, for how setting object of reference, guarantee that object of reference and tissue to be measured carry out testing process as for same time and spatial dimension, be that can object of reference at utmost guarantee to test correct precondition.With can with the polypeptide of antibody generation specific reaction as positive control, on same slide, react with to be measured organizing simultaneously, can successfully manage the risk of false wrong (like reagent configuration, reagent actual effect, the operating mistake of mistake) generation.
Can with the polypeptide of antibody generation specific reaction or albumen as positive control, have following several advantages: (1) can repeat, (2) can endlessly be supplied, (3) antigentic specificity, (4) cheapness can be produced in a large number, (5) are permanent to keep stable.
Description of drawings
Fig. 1 is a polypeptide point sample synoptic diagram; 1 expression label wherein first is arranged negative spot, is the bovine serum albumin(BSA) of 1 mg/mL; The positive spot of second row is respectively 0.001 mg/mL, 0.001 mg/mL, 0.01 mg/mL, 0.01 mg/mL concentration polypeptide from left to right; The positive spot of the 3rd row is respectively 0.1 mg/mL, 0.1 mg/mL, 1 mg/mL, 1 mg/mL concentration polypeptide from left to right.
Fig. 2 is an appearance synoptic diagram on the polypeptide; 1 expression label wherein first is arranged negative spot, is the bovine serum albumin(BSA) of 1 mg/mL; The positive spot of second row is respectively 0.001 mg/mL, 0.001 mg/mL concentration polypeptide from left to right; The positive spot of the 3rd row is respectively 0.01 mg/mL, 0.01 mg/mL concentration polypeptide from left to right.
Embodiment
A kind of preparation method who is used for the positive object of reference of SABC procedure quality prosecution of the present invention may further comprise the steps:
1) polypeptide marker: selection can with the polypeptide fragment of target antibody specific reaction; Adopt phosphate buffer (PBS) to be configured to the different dilution solution of series; Adopt automatic point sample instrument or the manual top that polypeptide is printed to slide respectively; Or adopting the mode that polypeptide is embedded in the paraffin mass section to place slide top, wherein negative spot is with the bovine serum albumin(BSA) replacement polypeptide of 1 mg/mL.Concrete peptide concentration is selected antibody adjustment for use according to different;
2) dewaxing and aquation: use the slide of the good polypeptide of a step mark, fish for the histotomy of having opened up sheet, as for the middle and lower part of slide.With slide placed at room temperature 60 minutes or 60 ℃ of constant temperature ovens in baking 20 minutes.Whole slide places xylene to soak 10 minutes, soaks 10 minutes behind the replacing xylene again; Soaked 5 minutes in the absolute ethyl alcohol, soaked 5 minutes in 95% ethanol, soaked 5 minutes in 75% ethanol.Washed 2-3 time each 5 minutes with PBS, use 3%H
2O
2(80% methyl alcohol) drips on slide, and room temperature left standstill 10 minutes, and deactivation endogenous oxidase, PBS were washed 2-3 time each 5 minutes.Attention: except that particularly pointing out, the immersion process in the following steps need immerse histotomy and polypeptide in the liquid simultaneously; (if adopt cell smear or cell climbing sheet, do not adopt the paraffin section mode, omit step 2));
3) antigen retrieval:, select one of following method to carry out antigen retrieval (tissue, cell adopt the antigen retrieval step with formaldehyde or formalin fixed) according to the requirement of different tissues antigen:
A, antigen hot repair in slide being immersed EDTA (ph9.0) or 0.01m sodium citrate buffer solution (ph6.0), are placed in the pressure cooker that adds water again, cover Stainless steel pot cover, and high pressure treated that pressure returns to normal pressure after 10 minutes, take out natural cooling.Or adopt the micro-wave oven heating means, to boiling, organization chip is put into outage, 5-10 minute at interval, 1-2 time repeatedly at heating 0.01 sodium citrate buffer (ph6.0) in the micro-wave oven.
B, enzymic digestion method 0.1% trypsase commonly used and 0.4% pepsin liquid.Preheating was 37 ℃ before trypsase used, and slide is immersed enzyme liquid, and digestion time is about 5-30 minute.The antigen that being applicable to is fixed covers: comprise Collagen, GFAP, Complement, Cytokeratin, CerB-2, LCA, LN etc.
4) cooled slide was washed 2-3 time each 5 minutes with PBS, dripped normal goats serum confining liquid, room temperature 20 minutes is got rid of unnecessary liquid, drips anti-50 μ l, 4 ℃ spend the night or 37 ℃ 1 hour; PBS wash 3 times each 2 minutes; Drip two anti-45-50 μ l, 37 ℃ 1 hour; PBS washed 3 times each 5 minutes, dripped DAB colour developing liquid then, and the DAB colour developing is 5-10 minute under the room temperature, grasps dye levels at microscopically.PBS or tap water flushing 10 minutes, dehydration, transparent, mounting, microscopy.
5) according to the change color of labeling polypeptide spot variable concentrations, as the standard of decision operation flow process and reagent; If spot Show Color and polypeptide show the trend of deepening gradually from low to high from concentration, description operation flow process and reagent are qualified; Through microscope histotomy is observed again,, judged according to the contrast of target position color on the histotomy and polypeptide spot colors.
The proportioning of described various solution is:
1.PBS damping fluid (pH7.2-7.4): NaC137mmol/L, KCl 2.7mmol/L, Na
2HPO
44.3mmol/L, KH
2PO
41.4 mmol/L;
2.0.01mol/L sodium citrate buffer solution (pH6.0,1000ml): trisodium citrate 3g, citric acid 0.4g;
3.0.5mol/L edta buffer liquid (pH9.0): dissolving 186.1g EDTA * 2H2O in the 700ml water, transfer to pH 9.0 with 10mmol/L NaOH, add deionized water to 1000ml;
4. enzymic digestion liquid: a. 0.1% trypsase: prepare with 0.1%CaCl 12 (pH 7.8).B.0.4% pepsin liquid: with the HCl preparation of 0.1mol/L;
5. 3% methyl alcohol-H
2O
2Solution: use 30% H
2O
2Prepare with 80% methanol solution.
Described polypeptide be configured to series different dilution solution, be meant affinity according to different antibody responses, configure can on slide, show series by shallow to dense color depth scope.Because different antibody response effects is variant,, need the peptide concentration of configuration different series therefore to different antibody;
Described polypeptide is can organize the anti-amino acid polymer that specific reaction can take place of used specificity one with detection through what screening obtained, and is consistent with one section sequence in target position albumen or the target position albumen in the tissue.
Below in conjunction with specific embodiment the present invention is carried out detailed explanation, following embodiment is in order to further specify the present invention, but should not be regarded as limiting the present invention.Below used biochemical reagents except that indicating especially all available from Sangon Biotech (Shanghai) Co., Ltd., antibody all steps neoformation scientific and technological development company available from Foochow.Clinical tissue paraffin section among the embodiment provides for Foochow steps neoformation scientific and technological development company.
Embodiment 1:
Ki67 phage polypeptide library adopts the PCR mode to make up.From stomach cancer cell HepG2 cell, extract total RNA, behind the reverse transcription, obtain the mRNA sequence of people Ki67 albumen from the NCBI website; According to the sequences Design primer; Adopting the cDNA sequence of pcr amplification Ki67, is template with the cDNA sequence that obtains then, increases with heminested PCR again.Primer after the amplification is used E
CoR1 and H
IndIII restriction endonuclease (available from Sangon Biotech (Shanghai) Co., Ltd.) enzyme is cut; According to guiding book in the NewEnglandBiolabs company phage display peptide library kit guidance; Be connected in the bacteriophage; Carry out polypeptide expression (all steps are all undertaken by guiding book in the New England Biolabs company phage display peptide library kit, and kit is available from U.S. New England Biolabs company)
Primer in the experiment (Sangon Biotech (Shanghai) Co., Ltd. is synthetic) sequence is as shown in the table:
Table 1 PCR the primer
Annotate: in above-mentioned table 1 primer, Forward adds E 5 '
CoR1 restriction enzyme site sequence (ACGAATTC), Reverse adds H 5 '
IndIII restriction enzyme site sequence (CGAAGCTT).
With the commercial Ki67 antibody 100-200 that obtains of PBS dilution doubly, on the 96 hole microwell plate plates, every hole adds 100 μ L; 4 ℃ encapsulate and spend the night; Sucking-off coating buffer, PBS are washed plate 3 times, and 3% BA solution is at 37 ℃ of sealing 2h; PBS washes plate 3 times, adds different clone's phage antibody library 100 μ L respectively and (contains 2 * 10 approximately
11CFU), hatch 1.5h for 37 ℃, wash plate 3 times (increasing by 1 time) by wheel with PBS (PBS that contains 0.5% Tween-20).With 100 μ L eluents (glycocoll-hydrochloric acid, pH2.2) wash-out is adsorbed on the bacteriophage in the enzyme mark hole, with 50 μ L Tris-HCl (1mol/L, pH8.0) in and eluate, get 10 μ L and be used for titer determination, all the other eluates are used for the next round elutriation after increasing.Phage titre under wash-out reaches 1 * 10
7CFU, this clone's peptide sequence after order-checking, synthesize this polypeptide as target sequence.
Polypeptide is diluted to the concentration of 1mg/mL, 0.1mg/mL, 0.01 mg/mL, 0.001 mg/mL respectively with PBS; Then on slide top near labelled position; Adopt wherein negative spot to replace polypeptide as negative control, four points of first row point with the bovine serum albumin(BSA) of 1 mg/mL; Second row and the 3rd row press peptide concentration order from low to high point sample successively, and each point is gone up appearance 0.1mL, two points of every kind of concentration.Obtain the array of four points of the three every rows of row after having put, topmost a row is as negative control, and second row and the 3rd row are as positive control.As shown in Figure 1.
Adopt histotome that the paraffin organization piece is cut into slices, the slide with polypeptide marker behind the aquation exhibition sheet drags for sheet, histotomy is placed on the middle and lower part of slide.According to the requirement of Ki67 first antibody (rabbit monoclonal antibody, below identical), carry out antigen retrieval.The requirement that Ki67 one heat resistanceheat resistant of this operation employing is repaired is immersed slide in EDTA (pH9.0) buffer solution, is placed in the pressure cooker that adds water, covers Stainless steel pot cover, and high pressure treated that pressure returns to normal pressure after 10 minutes, takes out natural cooling.Cooled slide was washed 2-3 time each 5 minutes with PBS, dripped normal goats serum confining liquid, room temperature 20 minutes is got rid of unnecessary liquid, drips anti-50 μ l, 4 ℃ spend the night or 37 ℃ 1 hour; PBS wash 3 times each 2 minutes; Drip goat-anti rabbit two anti-45-50 μ l, 37 ℃ 1 hour; PBS washed 3 times each 5 minutes, dripped DAB colour developing liquid then, and the DAB colour developing is 5-10 minute under the room temperature, grasps dye levels at microscopically.PBS or tap water flushing 10 minutes, dehydration, transparent, mounting, microscopy.According to polypeptide spot colour development difference, false positive reaction and misoperation have been judged whether.Do not have tangible chromogenic reaction like first row's spot, and two row's spot colors are significantly higher than first row down, color demonstrates variation from shallow to deep from low to high according to peptide concentration, shows that then the experimental implementation process is errorless.If first row's color reaction is consistent with second row, then the description operation process has false positive to occur, and the result is unreliable; If it is shallow that second row, the 3rd row do not develop the color or developed the color, show that then operation or reagent possibly go wrong.If spot shows normal, can be used as reference, the dyeing of pathological anatomy is analyzed.
Embodiment 2: buy the people source Ki67 albumen (available from Sigma company) that obtains through commercial sources; Be diluted to the concentration of 0.5mg/mL, 0.05mg/mL, 0.005 mg/mL, 0.0005 mg/mL respectively with PBS; Then on slide top near labelled position; Adopt the 1mg/mL bovine serum albumin(BSA) as negative control, four points of first row point; Second row and the 3rd row press peptide concentration order from low to high point sample successively, and each point is gone up appearance 0.1mL, two points of every kind of concentration.Obtain the array of four points of the three every rows of row after having put, topmost a row is as negative control, and second row and the 3rd row are as positive control.(point sample mode such as embodiment 2)
Get 10 routine doubtful lymphoma tissue paraffin specimens clinically, adopt histotome that the paraffin organization piece is cut into slices, the slide with polypeptide marker behind the aquation exhibition sheet drags for sheet, histotomy is placed on the middle and lower part of slide.According to the requirement of first antibody (clinical provide), carry out antigen retrieval with commercialization antibody.The requirement that Ki67 one heat resistanceheat resistant of this operation employing is repaired is immersed slide in EDTA (pH9.0) buffer solution, is placed in the pressure cooker that adds water, covers Stainless steel pot cover, and high pressure treated that pressure returns to normal pressure after 10 minutes, takes out natural cooling.Cooled slide was washed 2-3 time each 5 minutes with PBS, dripped normal goats serum confining liquid, room temperature 20 minutes is got rid of unnecessary liquid, drips anti-50 μ l, 4 ℃ spend the night or 37 ℃ 1 hour; PBS wash 3 times each 2 minutes; Drip two anti-45-50 μ l, 37 ℃ 1 hour; PBS washed 3 times each 5 minutes, dripped DAB colour developing liquid then, and the DAB colour developing is 5-10 minute under the room temperature, grasps dye levels at microscopically.PBS or tap water flushing 10 minutes, dehydration, transparent, mounting, microscopy.According to polypeptide spot colour development difference, false positive reaction and misoperation have been judged whether.Do not have tangible chromogenic reaction like first row's spot, and two row's spot colors are significantly higher than first row down, color demonstrates variation from shallow to deep from low to high according to peptide concentration, shows that then the experimental implementation process is errorless.If first row's color reaction is consistent with second row, then the description operation process has false positive to occur, and the result is unreliable; If it is shallow that second row, the 3rd row do not develop the color or developed the color, show that then operation or reagent possibly go wrong.If spot shows normal, can be used as reference, the dyeing of pathological anatomy is analyzed.If it is obviously painted that tissue does not have, then can be judged as negative tissue, no lymthoma generates, and is designated as (-); If structural dyed color is weaker than second row, then can be judged as low potential malignancy non-Hodgkin lymphomas, be designated as (+); If color is higher than the 3rd row, then can be judged as high malignancy non-Hodgkin lymphomas, be designated as (+++); If between between the two, can be judged as by the end to high stage of development, be designated as (++).Three rows' coloration result compares on the coloration result of 10 routine pathological tissues and the slide, and the result is consistent with pathology doctor result of determination.
Table 2 pathological tissue dyeing diagnostic result
Embodiment 3
Preparation method prepares the Ki67 polypeptide among the employing embodiment 1, and the concentration that polypeptide dilutes 0.01 mg/mL, 0.001 mg/mL respectively with PBS adopts the 1mg/mL bovine serum albumin(BSA) as negative control; In the polypeptide solution and bovine serum albumin solution of variable concentrations, adding final concentration is the agarose of 30g/L, is heated to after 70 ° of C melt fully; Suction is in the 1mL plastic injector; Be frozen into gel column under the room temperature, gel column taken out in the tip portion cutting-out of syringe, it is regarded tissue treatment with sharp blade; Through being placed on the wax the inside behind the SABC routine operation, then wax being cut into suitable size and being embedded in and be used for the microtome section in the paraffin mass.
As negative control, two points of first row point; Second row and the 3rd row add section by peptide concentration order from low to high, two points of every kind of concentration successively.Obtain the array of two points of the three every rows of row after having put, topmost a row is as negative control, and second row and the 3rd row are as positive control.As shown in Figure 2.
Get 10 routine doubtful lymphoma tissue paraffin specimens clinically, adopt histotome that the paraffin organization piece is cut into slices, the slide with polypeptide marker behind the aquation exhibition sheet drags for sheet, histotomy is placed on the middle and lower part of slide.According to the requirement of first antibody (clinical provide), carry out antigen retrieval with commercialization antibody.The requirement that Ki67 one heat resistanceheat resistant of this operation employing is repaired is immersed slide in EDTA (pH9.0) buffer solution, is placed in the pressure cooker that adds water, covers Stainless steel pot cover, and high pressure treated that pressure returns to normal pressure after 10 minutes, takes out natural cooling.Cooled slide was washed 2-3 time each 5 minutes with PBS, dripped normal goats serum confining liquid, room temperature 20 minutes is got rid of unnecessary liquid, drips anti-50 μ l, 4 ℃ spend the night or 37 ℃ 1 hour; PBS wash 3 times each 2 minutes; Drip two anti-45-50 μ l, 37 ℃ 1 hour; PBS washed 3 times each 5 minutes, dripped DAB colour developing liquid then, and the DAB colour developing is 5-10 minute under the room temperature, grasps dye levels at microscopically.PBS or tap water flushing 10 minutes, dehydration, transparent, mounting, microscopy.According to polypeptide spot colour development difference, false positive reaction and misoperation have been judged whether.Do not have tangible chromogenic reaction like first row's spot, and two row's spot colors are significantly higher than first row down, color demonstrates variation from shallow to deep from low to high according to peptide concentration, shows that then the experimental implementation process is errorless.If first row's color reaction is consistent with second row, then the description operation process has false positive to occur, and the result is unreliable; If it is shallow that second row, the 3rd row do not develop the color or developed the color, show that then operation or reagent possibly go wrong.If spot shows normal, can be used as reference, the dyeing of pathological anatomy is analyzed.If it is obviously painted that tissue does not have, then can be judged as negative tissue, no lymthoma generates, and is designated as (-); If structural dyed color is weaker than second row, then can be judged as low potential malignancy non-Hodgkin lymphomas, be designated as (+); If color is higher than the 3rd row, then can be judged as high malignancy non-Hodgkin lymphomas, be designated as (+++); If between between the two, can be judged as by the end to high stage of development, be designated as (++).Three rows' coloration result compares on the coloration result of 10 routine pathological tissues and the slide, and the result is consistent with pathology doctor result of determination.
Table 3 pathological tissue dyeing diagnostic result
Embodiment 4:
People source Ki67 albumen (available from Sigma company) through commercial sources is bought with the concentration that PBS dilutes 0.005 mg/mL, 0.0005 mg/mL respectively, adopts the 1mg/mL lowlenthal serum as negative control; In the polypeptide solution and bovine serum albumin solution of variable concentrations, adding final concentration is the agarose of 30g/L, is heated to after 70 ° of C melt fully; Suction is in the 1mL plastic injector; Be frozen into gel column under the room temperature, gel column taken out in the tip portion cutting-out of syringe, it is regarded tissue treatment with sharp blade; Through being placed on the wax the inside behind the SABC routine operation, then wax being cut into suitable size and being embedded in and be used for the microtome section in the paraffin mass.
As negative control, two points of first row point; Second row and the 3rd row add section by peptide concentration order from low to high, two points of every kind of concentration successively.Obtain the array of two points of the three every rows of row after having put, topmost a row is as negative control, and second row and the 3rd row are as positive control.Point sample mode such as embodiment 3.
Get 10 routine doubtful lymphoma tissue paraffin specimens clinically, adopt histotome that the paraffin organization piece is cut into slices, the slide with polypeptide marker behind the aquation exhibition sheet drags for sheet, histotomy is placed on the middle and lower part of slide.According to the requirement of first antibody (clinical provide), carry out antigen retrieval with commercialization antibody.The requirement that Ki67 one heat resistanceheat resistant of this operation employing is repaired is immersed slide in EDTA (pH9.0) buffer solution, is placed in the pressure cooker that adds water, covers Stainless steel pot cover, and high pressure treated that pressure returns to normal pressure after 10 minutes, takes out natural cooling.Cooled slide was washed 2-3 time each 5 minutes with PBS, dripped normal goats serum confining liquid, room temperature 20 minutes is got rid of unnecessary liquid, drips anti-50 μ l, 4 ℃ spend the night or 37 ℃ 1 hour; PBS wash 3 times each 2 minutes; Drip two anti-45-50 μ l, 37 ℃ 1 hour; PBS washed 3 times each 5 minutes, dripped DAB colour developing liquid then, and the DAB colour developing is 5-10 minute under the room temperature, grasps dye levels at microscopically.PBS or tap water flushing 10 minutes, dehydration, transparent, mounting, microscopy.According to polypeptide spot colour development difference, false positive reaction and misoperation have been judged whether.Do not have tangible chromogenic reaction like first row's spot, and two row's spot colors are significantly higher than first row down, color demonstrates variation from shallow to deep from low to high according to peptide concentration, shows that then the experimental implementation process is errorless.If first row's color reaction is consistent with second row, then the description operation process has false positive to occur, and the result is unreliable; If it is shallow that second row, the 3rd row do not develop the color or developed the color, show that then operation or reagent possibly go wrong.If spot shows normal, can be used as reference, the dyeing of pathological anatomy is analyzed.If it is obviously painted that tissue does not have, then can be judged as negative tissue, no lymthoma generates, and is designated as (-); If structural dyed color is weaker than second row, then can be judged as low potential malignancy non-Hodgkin lymphomas, be designated as (+); If color is higher than the 3rd row, then can be judged as high malignancy non-Hodgkin lymphomas, be designated as (+++); If between between the two, can be judged as by the end to high stage of development, be designated as (++).Three rows' coloration result compares on the coloration result of 10 routine pathological tissues and the slide, and the result is consistent with pathology doctor result of determination.
Table 4 pathological tissue dyeing diagnostic result
< 110>University of Fuzhou
< 120>a kind of preparation method who is used for the positive object of reference of SABC procedure quality prosecution
<160> 14
<170> PatentIn?version?3.3
<210> 1
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16
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ctccctctac?atctgctttc?ctgat
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cgcctggtta?ctatca
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gtgcccacca?aaggac
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agcgtcacgg?gaacca
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cgcctggtta?ctatca
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Claims (4)
1. preparation method who is used for the positive object of reference of SABC procedure quality prosecution; It is characterized in that; Comprise following process steps: can with the polypeptide or the albumen of antibody generation specific reaction, variable concentrations are set are adsorbed on the slide in advance, or the polypeptide or the albumen paraffin section that will pass through the variable concentrations of embedding are placed on the slide in advance; Experience routine immunization group step simultaneously with histopathologic slide, the coloration result of polypeptide or albumen is as the positive reference of SABC process.
2. the preparation method who is used for the positive object of reference of SABC procedure quality prosecution according to claim 1 is characterized in that, described polypeptide or albumen are the epi-position of tissue antigen in the histopathologic slide.
3. the preparation method who is used for the positive object of reference of SABC procedure quality prosecution according to claim 1; It is characterized in that; Described polypeptide or albumen are adsorbed on the slide in advance, are the modes with covalency or physisorption, are adsorbed on the slide that SABC uses.
4. the preparation method who is used for the positive object of reference of SABC procedure quality prosecution according to claim 1 is characterized in that, described coloration result, and the spot colors of polypeptide or albumen shows gradually from concentration from low to high deepens; Through microscope histopathologic slide is observed again, judged according to the contrast of the spot colors of target position color and polypeptide or albumen in the histopathologic slide.
Priority Applications (1)
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