CN103076448B - Preparation method and device for novel labeling technique of cervix cancer cells - Google Patents
Preparation method and device for novel labeling technique of cervix cancer cells Download PDFInfo
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- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 title claims abstract description 37
- 201000010881 cervical cancer Diseases 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 31
- 238000002372 labelling Methods 0.000 title claims abstract description 23
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 10
- 229940038773 trisodium citrate Drugs 0.000 claims description 10
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- 238000003756 stirring Methods 0.000 claims description 8
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- 239000000203 mixture Substances 0.000 claims description 7
- 239000012530 fluid Substances 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 4
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
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Abstract
The embodiment of the invention discloses a preparation method and device for a novel labeling technique of cervix cancer cells; and the method and the device can simply, quickly and accurately diagnose the cervix cancer cells. The method provided by the embodiment comprises the following steps: preparing colloidal gold; using the colloidal gold and antibodies for resisting epithelial cell adhesion molecules to prepare immunogold; and using the immunogold to label the epithelial cell adhesion molecules in pre-collected samples, and tabletting the samples. As colloidal gold particles are visible under a general optical microscope, when the epithelial cell adhesion molecules are labeled by using the colloidal gold and the immunogold which is prepared by the antibodies for resisting the epithelial cell adhesion molecules, the property of the epithelial cell adhesion molecules can be determined through direct observation; and as the expression level relevancy of the cervix cancer cells with the epithelial cell adhesion molecules on the surfaces of the cells is higher, the cervix cancer cells can be simply, quickly and accurately diagnosed through the preparation method and device in the embodiment of the invention.
Description
Technical field
The embodiment of the present invention relates to field of biomedicine technology, is specifically related to a kind of preparation method and device of labelling technique of new cervical cancer cell.
Background technology
Cervix cancer is one of modal malignant tumour, and the incidence of disease is positioned at the second of female tumor.This disease is died from the whole world every year nearly 200,000 women, and the early diagnosis and therapy of antithetical phrase precancerous lesions of uterine cervix can reduce its incidence.
A kind of cervical cancer cell Examined effect more advanced is in the world thinprep cytologic test (TCT at present, Thinprep Cytologic Test), TCT checks it is adopt liquid-based thin-layer cell detection system to detect cervical cell and carry out the method for cytology specification diagnosis, satisfaction and the abnormal cervical cell recall rate of sample is significantly improved compared with traditional cervical smear Pap smear inspection, can also find part precancerous lesion, infected by microbes is as mould, trichomonad, virus, Chlamydia etc. simultaneously.
But utilize thinprep cytologic test to carry out pathological diagnosis not only to need veteran expert, even and if like this, still there will be mistaken diagnosis and fail to pinpoint a disease in diagnosis.Therefore the technology developing easy quick detection cancer cell has very large clinical meaning.
Summary of the invention
Embodiments provide a kind of preparation method and device of labelling technique of new cervical cancer cell, antidiastole can be carried out to cervical cancer cell simply, fast and exactly.
The preparation method of the labelling technique of the new cervical cancer cell that the embodiment of the present invention provides, comprising:
Prepare collaurum;
The antibody of described collaurum and anti-epithelial cell adhesion molecule is utilized to prepare Immuno gold;
Described Immuno gold is utilized to mark the epithelial cell adhesion molecule in the sample of pre-collecting;
Film-making is carried out to described sample.
Alternatively, the preparation method of described collaurum comprises:
(1) gold chloride is mixed with the aqueous solution that concentration is 0.005%-0.015% and get 100-200ml be heated to boiling;
(2) stir aqueous solution of chloraurate, and add to described aqueous solution of chloraurate the trisodium citrate aqueous solution that 3-6ml concentration is 0.5-1.5%;
(3) 15-20 minute is boiled in the solution heating after step (2) process;
(4) volume of described solution after step (3) process is recovered to original volume with DDW in solution 2-10 minute of being cooled through after step (3) process to room temperature, obtains colloidal gold solution.
Alternatively, the described method utilizing the antibody of collaurum and anti-epithelial cell adhesion molecule to prepare Immuno gold comprises:
(1) pH value of described colloidal gold solution is regulated to be 7.5 to 8.5;
(2) antibody of described anti-epithelial cell adhesion molecule is inserted put into DDW after in bag filter and dialyse 2-4 hour or spend the night;
(3) centrifugal force is carried out at 4 DEG C to the solution after step (2) process and be centrifugation 50-70 minute of 50000-100000g and the polymkeric substance removed in described solution;
(4) supernatant of solution after step of learning from else's experience (3) process, the protein adjusting described supernatant to 0.5-1mg/ml concentration and using the antibody storage liquid of the supernatant after adjustment as anti-epithelial cell adhesion molecule;
(5) after the antibody storage liquid of described anti-epithelial cell adhesion molecule being made serial dilution with the borate buffer solution of concentration to be 0.004-0.006mol/l pH value be 8.5-9.5, the antibody serial dilutions of getting the anti-epithelial cell adhesion molecule of 0.05-0.15ml is added in colloidal gold solution described in 0.5-1.5ml, a pipe is separately established not add the control tube of the antibody serial dilutions of described anti-epithelial cell adhesion molecule, respectively to adding the NaCl solution that 0.05-0.15ml concentration is 8-12% in each pipe after 4-6 minute, 1.5-2.5 hour is left standstill after mixing the solution of each pipe, can make the stable protein content of collaurum adds 9%-11% again and described protein content is designated as optimum mark protein content,
(6) adjust protein content content in described supernatant be described optimum mark protein content and add described colloidal gold solution and fully mix 15-30 minute;
(7) in the solution after step (6) process, adding concentration is that the PEG-2000 of 2%-4% makes the PEG-2000 ultimate density of described solution be 0.05%-0.1% and stirs described solution 10-15 minute;
(8) sediment fraction in the solution after step of learning from else's experience (7) process is also with the NaN of BSA and 0.01%-0.03% containing 0.5%-1.5%
3the damping fluid of concentration to be 0.01-0.03mol/l pH value the be Tris-HCl of 7.0-7.4 dilute described sediment fraction, obtain Immuno gold;
(9) reclaim described Immuno gold and carry out filtration sterilization with miillpore filter.
Alternatively, utilize described in step described Immuno gold the epithelial cell adhesion molecule in the sample of pre-collecting marked after and also comprise before film-making being carried out to sample described in step:
Pre-service is carried out to slide;
The antibody of sample cell is hatched;
Describedly pre-service carried out to slide comprise:
Slide described in the alcohol immersion process being 93%-97% by concentration;
Described slide is cleaned with DDW;
By 3-aminopropyl-3-(ethoxymethyl) silane package by slide.
Alternatively, also comprise after film-making being carried out to sample described in step:
Microscope is utilized to observe described film-making.
Alternatively, also comprise after film-making being carried out to sample described in step:
Described film-making is dyeed;
Microscope is utilized to observe described film-making.
The preparation facilities of the labelling technique of the new cervical cancer cell that the embodiment of the present invention provides, comprising:
Prepare unit one, for the preparation of collaurum;
Preparing unit two, preparing Immuno gold for utilizing the antibody of described collaurum and anti-epithelial cell adhesion molecule;
Indexing unit, marks the epithelial cell adhesion molecule in the sample of pre-collecting for utilizing described Immuno gold;
Film-making unit, for carrying out film-making to described sample.
Alternatively, described device also comprises:
Pretreatment unit, for carrying out pre-service to slide;
Hatch unit, for hatching the antibody of sample cell.
In the embodiment of the present invention, first prepare collaurum; Then the antibody of described collaurum and anti-epithelial cell adhesion molecule is utilized to prepare Immuno gold; Then described Immuno gold is utilized to mark the epithelial cell adhesion molecule in the sample of pre-collecting; Finally film-making is carried out to described sample.Because colloid gold particle is visible under ordinary optical microscope, when the Immuno gold utilizing the antibody of collaurum and anti-epithelial cell adhesion molecule to prepare goes to mark epithelial cell adhesion molecule, by directly observing the character just can determining epithelial cell adhesion molecule, because the expression degree of correlation of the epithelial cell adhesion molecule on cervical cancer cell and its surface is very high, so antidiastole can be carried out to cervical cancer cell simply, fast and exactly.
Accompanying drawing explanation
Fig. 1 is labelling technique of cervical cancer cell new in the embodiment of the present invention and preparation method thereof first embodiment process flow diagram;
Fig. 2 is labelling technique of cervical cancer cell new in the embodiment of the present invention and preparation method thereof second embodiment process flow diagram;
Fig. 3 is labelling technique and the preparation facilities example structure figure thereof of cervical cancer cell new in the embodiment of the present invention.
Embodiment
Embodiments provide a kind of preparation method and device of labelling technique of new cervical cancer cell, antidiastole can be carried out to cancer cell and mesothelial cell simply, fast and exactly.
Refer to Fig. 1, first embodiment of the preparation method of the labelling technique of cervical cancer cell new in the embodiment of the present invention comprises:
101, collaurum is prepared;
The composition mainly gold chloride of collaurum, preparation method can adopt trisodium citrate reduction method, and the reductive agent usually adopted has trisodium citrate, tannic acid, ascorbic acid, white phosphorus and sodium borohydride.
102, the antibody of collaurum and anti-epithelial cell adhesion molecule is utilized to prepare Immuno gold;
After preparing collaurum, the antibody of collaurum and anti-epithelial cell adhesion molecule can be utilized to prepare Immuno gold.
103, Immuno gold is utilized to mark the epithelial cell adhesion molecule in the sample of pre-collecting;
After preparing Immuno gold, Immuno gold can be utilized to mark the epithelial cell adhesion molecule in the sample of pre-collecting.
104, film-making is carried out to sample.
Utilize after Immuno gold marks the epithelial cell adhesion molecule in the sample of pre-collecting, film-making can be carried out to sample.
In the embodiment of the present invention, first prepare collaurum; Then the antibody of collaurum and anti-epithelial cell adhesion molecule is utilized to prepare Immuno gold; Then Immuno gold is utilized to mark the epithelial cell adhesion molecule in the sample of pre-collecting; Finally film-making is carried out to sample.Because colloid gold particle is visible under ordinary optical microscope, when the Immuno gold utilizing the antibody of collaurum and anti-epithelial cell adhesion molecule to prepare goes to mark epithelial cell adhesion molecule, by directly observing the character just can determining epithelial cell adhesion molecule, because the expression degree of correlation of the epithelial cell adhesion molecule on cervical cancer cell and its surface is very high, so antidiastole can be carried out to cervical cancer cell simply, fast and exactly.
Simply describe first embodiment of the preparation method of the labelling technique of the new cervical cancer cell of the present invention above, below second embodiment of the preparation method of the labelling technique of the new cervical cancer cell of the present invention is described in detail, refer to Fig. 2, second embodiment of the preparation method of the labelling technique of cervical cancer cell new in the embodiment of the present invention comprises:
201, collaurum is prepared;
The composition mainly gold chloride of collaurum, preparation method can adopt trisodium citrate reduction method, and the reductive agent usually adopted has trisodium citrate, tannic acid, ascorbic acid, white phosphorus and sodium borohydride.Principle and concrete operation method as follows:
2HAuCl
4+3C
6H
8O
7=2Au+3C
5H
6O
5+8HCl+3CO
2
The concrete grammar preparing collaurum comprises: by gold chloride (HAuCl
4) to be mixed with concentration be 0.005%-0.015%, can be the aqueous solution of 0.01% and get 100-200ml, can be heated to boil for 100ml; Stir aqueous solution of chloraurate, and add 3-6ml to aqueous solution of chloraurate, can be 5ml, concentration be 0.5-1.5%, can be the trisodium citrate aqueous solution of 1%; Then continuing heating and boil above-mentioned solution 15-20 minute, can be 15 minutes; Finally solution obtained above is carried out cooling 2-10 minute, can be return to original volume with DDW after 5 minutes to room temperature, can collaurum be obtained.
202, the antibody of collaurum and anti-epithelial cell adhesion molecule is utilized to prepare Immuno gold;
After preparing collaurum, the antibody of collaurum and anti-epithelial cell adhesion molecule can be utilized to prepare Immuno gold.
The method specifically preparing Immuno gold can be: can be first the K of 0.2mol/l by concentration
2cO
3or concentration is that the HCl of 0.1mol/l regulates the pH of colloidal gold solution to set point value, can be 7.5 to 8.5; Then the antibody of anti-epithelial cell adhesion molecule is inserted after in bag filter, put into DDW dialyse 2-4 hour or spend the night, then above-mentioned solution being carried out at 4 DEG C centrifugal force is 50000-100000g, can be the centrifugation 50-70 minute of 100000g, can be 60 minutes and remove the polymkeric substance in described solution, get the supernatant of above-mentioned solution, the protein adjusting described supernatant to 0.5-1mg/ml concentration and using the antibody storage liquid of above-mentioned supernatant as anti-epithelial cell adhesion molecule.
After obtaining the antibody storage liquid of anti-epithelial cell adhesion molecule, be 0.004-0.006mol/l by concentration, can be 0.005mol/l, basicity is 8.5-9.5, can be 9.0 borate buffer solution the antibody storage liquid of anti-epithelial cell adhesion molecule is made serial dilution after, get 0.05-0.15ml, 0.5-1.5ml can be added to for the antibody serial dilutions of the anti-epithelial cell adhesion molecule of 0.1ml, can be in 1ml colloidal gold solution, separately establish a control tube not adding the antibody matter serial dilutions of anti-epithelial cell adhesion molecule, 4-6 minute, can be add 0.05-0.15ml respectively in each test tube after 5 minutes, can be 8-12% for 0.1ml concentration, it can be the NaCl solution of 10%, 1.5-2.5 hour is left standstill after mixing the solution of each pipe, it can be 2 hours, can make the stable protein content of collaurum adds 9%-11% again, can be 10% and above-mentioned protein content is designated as optimum mark protein content.
After obtaining optimum mark protein content, in adjustment supernatant, protein content content is optimum mark protein content, get above-mentioned supernatant add colloidal gold solution and fully mix 15-30 minute, it can be 15 minutes, then adding concentration is 2%-4%, can be 3% PEG-2000 make the PEG-2000 ultimate density in above-mentioned solution be 0.05%-0.1%, can be 0.05%, and stir above-mentioned solution 10-15 minute, sample thief Aspirate supernatant, then get sediment fraction at the bottom of pipe, with containing 0.5%-1.5%, can be BSA and 0.01%-0.03% of 1%, can be the NaN of 0.02%
3concentration be 0.01-0.03mol/l, can be 7.0-7.4 for 0.02mol/lpH value, can be that the damping fluid of the Tris-HCl of 7.2 dilutes above-mentioned sediment fraction, obtain Immuno gold, the yield of final Immuno gold be 10% of original volume.
Then can also be degerming with the filtering with microporous membrane of 0.22um, packing, and save backup at 4 DEG C.
203, Immuno gold is utilized to mark the epithelial cell adhesion molecule in the sample of pre-collecting;
After preparing Immuno gold, Immuno gold can be utilized to mark the epithelial cell adhesion molecule in the sample of pre-collecting.
204, pre-service is carried out to slide;
Above-mentionedly pre-service is carried out to slide can comprise: being first 93%-97% by concentration, can be the alcohol immersion process slide of 95%; Then slide is cleaned with DDW; Finally can be completed pre-service to slide by 3-aminopropyl-3-(ethoxymethyl) silane package by slide, above-mentioned preprocessing process can be set in step 206 before arbitrary step before or after, and do not limit with the present embodiment in after step 203.
205, the antibody of sample cell is hatched;
The above-mentioned process of hatching specifically can be, by the antibody of collaurum bag quilt and sample incubation at 35-39 DEG C, can be in the constant-temperature incubation case of 37 DEG C, reaction a period of time, can be 2 hours or spend the night in the constant-temperature incubation case of 4 DEG C, then using cleaning fluid successively after taking out, can be the PBS cleaning of 1%Tween20 by concentration, finally at rotating speed, can be centrifugal several times in the hydro-extractor of 1000rpm, can be 3 times, each continued for some time can be 5 minutes.
206, film-making is carried out to sample.
After complete paired samples cell carries out antibody incubation, film-making can be carried out to sample.Concrete flaking method is content of the prior art, repeats no more herein.
After film-making is carried out to described sample, can observe above-mentioned film-making.Before above-mentioned film-making is observed, can also dye to described film-making, and then observe.
In the embodiment of the present invention, first prepare collaurum; Then the antibody of collaurum and anti-epithelial cell adhesion molecule is utilized to prepare Immuno gold; Then Immuno gold is utilized to mark the epithelial cell adhesion molecule in the sample of pre-collecting; Finally film-making is carried out to sample.Wherein can also carry out pre-service to slide and antibody incubation be carried out to sample cell.Because colloid gold particle is visible under ordinary optical microscope, when the Immuno gold utilizing the antibody of collaurum and anti-epithelial cell adhesion molecule to prepare goes to mark epithelial cell adhesion molecule, by directly observing the character just can determining epithelial cell adhesion molecule, because the expression degree of correlation of the epithelial cell adhesion molecule on cervical cancer cell and its surface is very high, so antidiastole can be carried out to cervical cancer cell simply, fast and exactly.
Above the second embodiment of the labelling technique of the new cervical cancer cell of the present invention and preparation method thereof is described in detail, particularly prepare the process of collaurum and Immuno gold, introduce labelling technique and the preparation facilities embodiment thereof of the new cervical cancer cell of the present invention below, refer to Fig. 3, labelling technique and the preparation facilities embodiment thereof of cervical cancer cell new in the embodiment of the present invention comprise:
Prepare unit 1, for the preparation of collaurum;
Preparing unit 2 302, preparing Immuno gold for utilizing the antibody of collaurum and anti-epithelial cell adhesion molecule;
Indexing unit 303, marks the epithelial cell adhesion molecule in the sample of pre-collecting for utilizing Immuno gold;
Film-making unit 304, for carrying out film-making to sample.
Described device also comprises:
Pretreatment unit 305, for carrying out pre-service to slide;
Hatch unit 306, for hatching the antibody of sample cell.
First prepare unit 1 and prepare collaurum, the composition mainly gold chloride of collaurum, preparation method can adopt trisodium citrate reduction method, and the reductive agent usually adopted has trisodium citrate, tannic acid, ascorbic acid, white phosphorus and sodium borohydride.Principle and concrete operation method as follows:
2HAuCl
4+3C
6H
8O
7=2Au+3C
5H
6O
5+8HCl+3CO
2
The concrete grammar preparing collaurum comprises: by gold chloride (HAuCl
4) to be mixed with concentration be 0.005%-0.015%, can be the aqueous solution of 0.01% and get 100-200ml, can be heated to boil for 100ml; Stir aqueous solution of chloraurate, and add 3-6ml to aqueous solution of chloraurate, can be 5ml, concentration be 0.5-1.5%, can be the trisodium citrate aqueous solution of 1%; Then continuing heating and boil above-mentioned solution 15-20 minute, can be 15 minutes; Finally solution obtained above is carried out cooling 2-10 minute, can be return to original volume with DDW after 5 minutes to room temperature, can collaurum be obtained.
Prepare after unit 1 prepares collaurum, prepare unit 2 302 and the antibody of collaurum and anti-epithelial cell adhesion molecule can be utilized to prepare Immuno gold.
Preparing the method that unit 2 302 specifically prepares Immuno gold can be: can be first the K of 0.2mol/l by concentration
2cO
3or concentration is that the HCl of 0.1mol/l regulates the pH of colloidal gold solution to set point value, can be 7.5 to 8.5; Then the antibody of anti-epithelial cell adhesion molecule is inserted after in bag filter, put into DDW dialyse 2-4 hour or spend the night, then above-mentioned solution being carried out at 4 DEG C centrifugal force is 50000-100000g, can be the centrifugation 50-70 minute of 100000g, can be 60 minutes and remove polymkeric substance in described solution, get the supernatant of above-mentioned solution, adjustment supernatant protein to 0.5-1mg/ml concentration and using the antibody storage liquid of above-mentioned supernatant as anti-epithelial cell adhesion molecule.
After obtaining the antibody storage liquid of anti-epithelial cell adhesion molecule, be 0.004-0.006mol/l by concentration, can be 0.005mol/l, basicity is 8.5-9.5, can be 9.0 borate buffer solution the antibody storage liquid of anti-epithelial cell adhesion molecule is made serial dilution after, get 0.05-0.15ml, 0.5-1.5ml can be added to for the antibody serial dilutions of the anti-epithelial cell adhesion molecule of 0.1ml, can be in 1ml colloidal gold solution, separately establish a control tube not adding the antibody serial dilutions of anti-epithelial cell adhesion molecule, 4-6 minute, can be add 0.05-0.15ml respectively in each test tube after 5 minutes, can be 8-12% for 0.1ml concentration, it can be the NaCl solution of 10%, 1.5-2.5 hour is left standstill after mixing the solution of each pipe, it can be 2 hours, can make the stable protein content of collaurum adds 9%-11% again, can be 10% and above-mentioned protein content is designated as optimum mark protein content.
After obtaining optimum mark protein content, in adjustment supernatant, protein content content is optimum mark protein content, get above-mentioned supernatant add colloidal gold solution and fully mix 15-30 minute, it can be 15 minutes, then adding concentration is 2%-4%, can be 3% PEG-2000 make the PEG-2000 ultimate density of described solution be 0.05%-0.1%, can be 0.05%, and stir described solution 10-15 minute, sample thief Aspirate supernatant, then get sediment fraction at the bottom of pipe, with containing 0.5%-1.5%, can be BSA and 0.01%-0.03% of 1%, can be the NaN of 0.02%
3concentration be 0.01-0.03mol/l, can be 7.0-7.4 for 0.02mol/lpH value, can be that the damping fluid of the Tris-HCl of 7.2 dilutes above-mentioned sediment fraction, obtain colloid gold label compound, the yield of final colloid gold label compound be 10% of original volume.
Then can also be degerming with the filtering with microporous membrane of 0.22um, packing, and save backup at 4 DEG C.
Prepare after unit 2 302 prepares Immuno gold, indexing unit 303 can utilize Immuno gold to mark the epithelial cell adhesion molecule in the sample of pre-collecting, indexing unit 303 complete utilize Immuno gold the epithelial cell adhesion molecule in the sample of pre-collecting is marked after pretreatment unit 305 can carry out pre-service to slide.
Above-mentionedly pre-service is carried out to slide can comprise: being first 93%-97% by concentration, can be the alcohol immersion process slide of 95%; Then slide is cleaned with DDW; Finally can be completed pre-service to slide by 3-aminopropyl-3-(ethoxymethyl) silane package by slide.
After pretreatment unit 305 completes slide preprocessing process, hatching unit 306 can hatch the antibody of sample cell.
The above-mentioned process of hatching specifically can be, by the antibody of collaurum bag quilt and sample incubation at 35-39 DEG C, can be in the constant-temperature incubation case of 37 DEG C, reaction a period of time, can be 2 hours or spend the night in the constant-temperature incubation case of 4 DEG C, then using cleaning fluid successively after taking out, can be the PBS cleaning of 1%Tween20 by concentration, finally at rotating speed, can be centrifugal several times in the hydro-extractor of 1000rpm, can be 3 times, each continued for some time can be 5 minutes.
Hatch after process that unit 306 pairs of sample cells carry out antibody incubation completes, film-making unit 304 carries out film-making to described sample.Concrete flaking method is content of the prior art, repeats no more herein.
Film-making unit 304 pairs of samples can be observed above-mentioned film-making after carrying out film-making.Before above-mentioned film-making is observed, can also dye to film-making, and then observe.
In the embodiment of the present invention, prepare unit 1 and first prepare collaurum; Then preparing unit 2 302 utilizes the antibody of collaurum and anti-epithelial cell adhesion molecule to prepare Immuno gold; Then indexing unit 303 utilizes Immuno gold to mark the epithelial cell adhesion molecule in the sample of pre-collecting; Last film-making unit 304 pairs of samples carry out film-making.Wherein pretreatment unit 305 can also carry out pre-service to slide and hatch unit 306 pairs of sample cells carrying out antibody incubation.Because colloid gold particle is visible under ordinary optical microscope, when the Immuno gold utilizing the antibody of collaurum and anti-epithelial cell adhesion molecule to prepare goes to mark epithelial cell adhesion molecule, by directly observing the character just can determining epithelial cell adhesion molecule, because the expression degree of correlation of the epithelial cell adhesion molecule on cervical cancer cell and its surface is very high, so antidiastole can be carried out to cervical cancer cell simply, fast and exactly.
One of ordinary skill in the art will appreciate that all or part of step realized in above-described embodiment method is that the hardware that can carry out instruction relevant by program completes, described program can be stored in a kind of computer-readable recording medium, the above-mentioned storage medium mentioned can be ROM (read-only memory), disk or CD etc.
Above the preparation method of the labelling technique of a kind of new cervical cancer cell provided by the present invention and device are described in detail, for one of ordinary skill in the art, according to the thought of the embodiment of the present invention, all will change in specific embodiments and applications, in sum, this description should not be construed as limitation of the present invention.
Claims (4)
1. a preparation method for the labelling technique of new cervical cancer cell, is characterized in that, comprising:
Prepare collaurum;
The antibody of described collaurum and anti-epithelial cell adhesion molecule is utilized to prepare Immuno gold;
Described Immuno gold is utilized to mark the epithelial cell adhesion molecule in the sample of pre-collecting;
Film-making is carried out to described sample;
The preparation method of described collaurum comprises:
(1) gold chloride is mixed with the aqueous solution that concentration is 0.005%-0.015% and get 100-200ml be heated to boiling;
(2) stir aqueous solution of chloraurate, and add to described aqueous solution of chloraurate the trisodium citrate aqueous solution that 3-6ml concentration is 0.5-1.5%;
(3) 15-20 minute is boiled in the solution heating after step (2) process;
(4) volume of described solution after step (3) process is recovered to original volume with DDW in solution 2-10 minute of being cooled through after step (3) process to room temperature, obtains colloidal gold solution;
The described method utilizing the antibody of collaurum and anti-epithelial cell adhesion molecule to prepare Immuno gold comprises:
(1) pH value of described colloidal gold solution is regulated to be 7.5 to 8.5;
(2) antibody of described anti-epithelial cell adhesion molecule is inserted put into DDW after in bag filter and dialyse 2-4 hour or spend the night;
(3) centrifugal force is carried out at 4 DEG C to the solution after step (2) process and be centrifugation 50-70 minute of 50000-100000g and the polymkeric substance removed in described solution;
(4) supernatant of solution after step of learning from else's experience (3) process, the protein adjusting described supernatant to 0.5-1mg/ml concentration and using the antibody storage liquid of the supernatant after adjustment as anti-epithelial cell adhesion molecule;
(5) after the antibody storage liquid of described anti-epithelial cell adhesion molecule being made serial dilution with the borate buffer solution of concentration to be 0.004-0.006mol/l pH value be 8.5-9.5, the antibody serial dilutions of getting the anti-epithelial cell adhesion molecule of 0.05-0.15ml is added in colloidal gold solution described in 0.5-1.5ml, separately establish a control tube not adding the antibody serial dilutions of described anti-epithelial cell adhesion molecule, respectively to adding the NaCl solution that 0.05-0.15ml concentration is 8-12% in each pipe after 4-6 minute, 1.5-2.5 hour is left standstill after mixing the solution of each pipe, can make the stable protein content of collaurum adds 9%-11% again and described protein content is designated as optimum mark protein content,
(6) adjust protein content content in described supernatant be described optimum mark protein content and add described colloidal gold solution and fully mix 15-30 minute;
(7) in the solution after step (6) process, adding concentration is that the PEG-2000 of 2%-4% makes the PEG-2000 ultimate density of described solution be 0.05%-0.1% and stirs described solution 10-15 minute;
(8) sediment fraction in the solution after step of learning from else's experience (7) process is also with the NaN of BSA and 0.01%-0.03% containing 0.5%-1.5%
3the damping fluid of concentration to be 0.01-0.03mol/l pH value the be Tris-HCl of 7.0-7.4 dilute described sediment fraction, obtain Immuno gold;
(9) reclaim described Immuno gold and carry out filtration sterilization with miillpore filter.
2. the preparation method of the labelling technique of new cervical cancer cell according to claim 1, it is characterized in that, utilize described in step described Immuno gold the epithelial cell adhesion molecule in the sample of pre-collecting marked after and also comprise before film-making being carried out to sample described in step:
Pre-service is carried out to slide;
The antibody of sample cell is hatched;
Describedly pre-service carried out to slide comprise:
Slide described in the alcohol immersion process being 93%-97% by concentration;
Described slide is cleaned with DDW;
By 3-aminopropyl-3-(ethoxymethyl) silane package by slide.
3. the preparation method of the labelling technique of new cervical cancer cell according to claim 2, is characterized in that, also comprises after carrying out film-making to sample described in step:
Microscope is utilized to observe described film-making.
4. the preparation method of the labelling technique of new cervical cancer cell according to claim 2, is characterized in that, also comprises after carrying out film-making to sample described in step:
Described film-making is dyeed;
Microscope is utilized to observe described film-making.
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