CN102435735B - Immunohistochemical staining detection kit for colorectal cancer and application thereof - Google Patents
Immunohistochemical staining detection kit for colorectal cancer and application thereof Download PDFInfo
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- CN102435735B CN102435735B CN201110301990.9A CN201110301990A CN102435735B CN 102435735 B CN102435735 B CN 102435735B CN 201110301990 A CN201110301990 A CN 201110301990A CN 102435735 B CN102435735 B CN 102435735B
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Abstract
The invention discloses an immunohistochemical staining detection kit for the colorectal cancer and an application thereof. The kit comprises: a, an antihuman Interleukin-17F (IL-17F) antibody from rabbit; b, biotin-labeled goat anti-rabbit IgG; c, avidin-labeled HRP; d, a normal goat serum sealant; e, 3 V% H2O2; f, a DAB coloring solution; and g, hematorylin. The detection kit which has the characteristics of high sensitivity, high specificity and high accuracy can distinguish cancerous tissues from normal tissues and also can distinguish cancerous cells from normal cells in the cancerous tissues. The IL-17F positively expresses staining in epithelial cells of normal colon tissues, but negatively expresses in the epithelial cells of the cancerous cells. The detection kit which can effectively distinguish the epithelial cells of the colorectal cancer from the normal cells provides references for the clinical diagnosis and the clinical treatment.
Description
Technical field
The invention belongs to biological technical field, more specifically relate to a kind of immunohistochemical staining detection kit of colorectal cancer, also relate to a kind of purposes of the detection kit of the immunohistochemical staining for colorectal cancer simultaneously.
Background technology
Colorectal cancer is one of modal malignant tumor of digestive tract, accounts for the 3rd of gastroenteric tumor.The incidence and mortality of China, along with the raising of living standards of the people, the change of dietary structure, be the trend risen year by year.Colorectal cancer has become the disease that threatens human life and health can not be ignored.Colorectal cancer onset concealment, early symptom is not obvious, many patients when making a definite diagnosis in late period.When about 25% patient goes to a doctor for the first time, just shift, in addition, 40%-50% newly diagnoses the patient will continue the development appearance and shifts.Only being less than 5% patient can survive more than 5 years.Therefore, deeply to the fundamental research of colorectal cancer with improve the diagnosis of colorectal cancer, to the survival rate that improves knot rectum patient, reduce its case fatality rate and there is considerable clinical meaning.
The method of the current diagnosis for colorectal cancer comprises fecal occult blood testing, endoscopy, iconography procuratorial work, Bio-molecular analysis and histopathologic examination.In the diagnosis of colorectal carcinoma technology, different detection methods is that never ipsilateral has reflected the shape of tumor that is different from normal structure in various degree at present.Single detection methods can not the clear and definite state of an illness, need to, in conjunction with actual conditions, select multiple detection methods just can judge.
Wherein pathological diagnosis is also diagnosis the most reliably in diagnosis of colorectal carcinoma, is to clarify a diagnosis to draft the necessary foundation of therapeutic scheme.Conventional Pathomorphology inspection comprises exfoliative cytology inspection and biopsy.Because cast-off cells in the knot rectum often have sex change in various degree, negative findings can not be negated the existence of tumour, and the colorectal cancer exfoliative cytology checks that range of application is very limited clinically, mainly relies on histopathological examination to clarify a diagnosis.
SABC is the emerging technology developed rapidly over nearest more than 10 years.It is extensively used tumor research and diagnosis, its principle is to utilize antigen and the specific binding of antibody to react to detect unknown antigen or the antibody in tissue, mainly tumor associated antigen (Tumor Differentiation antigen and tumour embryonal antigen), so as to source and the differentiation degree of judgement tumour, assist pathological diagnosis and the antidiastole of tumour.Colouring method commonly used has the PAP method at present, be PAP method (peroxidaseantiperoxidase technique) and avidin-avidin-biotin complex method, i.e. ABC method (avidin-biotin-peroxidase complex technique).Utilize immunohistochemical method can be difficult to judge that the tumour in its source is differentiated to many conventional methods.For example detect the intermediate filament (intermediate filament) of cytoskeleton, its diameter average out to 10nm, between microtubule and microfilament.Intermediate filament has five classes: i.e. nerve fibril, glial fibrillary acidic protein, desmin (desmin), vimentin (vimentin) and keratin (keratin).They respectively have biological chemistry and immunological characteristic, and be present in respectively in dissimilar cell, therefore there is relative specificity, can be used to assist the tumour in the corresponding neurocyte of diagnosis, Deiter's cells, striated muscle and smooth muscle, mesenchymal tissue and epithelial cell source.At present can be too numerous to enumerate for the antibody of tumour auxiliary diagnosis and antidiastole.
Summary of the invention
The objective of the invention is to be to provide a kind of immunohistochemical staining detection kit of colorectal cancer, this antibody test has high sensitivity, the characteristics of high specific and high accuracy, not only cancerous issue and normal structure can be distinguished, cell and the normal cell of canceration in cancerous issue can also be distinguished.IL-17F is positive expression dyeing in normal colonic tissue's epithelial cell, and the expression that is negative in the cancerous tumor cell epithelium.
Another object of the present invention is to be to provide the application of a kind of detection kit of the immunohistochemical staining for colorectal cancer in investigation of clinical epidemiology, effectively distinguish epithelial cell and normal cell that colorectal cancer becomes, for clinical diagnosis and treatment provide reference frame.
To achieve these goals, the present invention adopts following technical measures:
A kind of immunohistochemical staining detection kit of colorectal cancer, this immunohistochemical staining kit contains: a) antibody of the anti-human Interleukin-17F (IL-17F) in rabbit source, b) biotin labeled goat anti-rabbit igg; C) HRP of Avidin mark; D) normal goats serum confining liquid; E) 3% (volume ratio) H
2o
2; F) DAB nitrite ion; G) haematoxylin.This kit using the low cell factor of expressing of this specificity in the cancerous issue of colorectal cancer patients of IL-17F as for detection of reference index.
Key is the antibody preparation of the anti-human Interleukin-17F (IL-17F) in rabbit source in the present invention, and its method comprises the following steps:
A) build the IL-17F expression vector.Kit
purchased from invitrogen company.The Fc fragment of people's Immunoglobulin IgG1 (University of Washington's Edward doctor A.Clark friendship is given) label is cloned in the pMT/BiP/V5-HisA expression plasmid provided in kit, obtains fruit bat expression system-immunoglobulin (Ig) fusion plasmid (DES-Ig).The cDNA sequence of method amplification human il-17 F by PCR, be cloned into the DES-Ig plasmid.
B) drosophila cell of setting up stable transfection IL-17F expression vector is S2 (cell provided in kit), copper sulphate inducible protein secreting, expressing purifying IL-17F.According to the kit instructions, by the pCoHygro plasmid co-transfection drosophila cell provided in the above-mentioned carrier successfully constructed and kit, be S2, screening obtains stable cell lines; And with the secreting, expressing of copper sulphate inducible protein.Collect supernatant, use albumin A-agarose (Protein A-Sepharose) pillar purchased from the enriching and purifying IL-17F-Ig of sigma company fusion.
C) immune rabbit obtains the polyclonal antibody of IL-17F and identifies.Concrete steps are that to get the 200ug immunogene be that IL-17F-Ig fusion and Freund's complete adjuvant carry out being mixed to form emulsion at 1: 1, are injected into the subcutaneous and rear leg muscle of rabbit both shoulders; The 2 weeks booster immunizations in interval, immune commercial weight reduces by half, and is mixed into the emulsion injection with incomplete Freunds adjuvant and closes on position; 2 weeks booster immunizations in interval again; Within the 7th day, get blood after injection.Draw serum, centrifugal, packing, frozen.Detect antibody titer.
Utilize the specific polyclonal antibody of anti-IL-17F in the present invention, conventional reagent (the biotin labeled goat anti-rabbit igg of combined immunization histochemical stain; The HRP of Avidin mark; Normal goats serum confining liquid; 3%H
2o
2; The DAB nitrite ion; Haematoxylin), the pathology detection of convenient, efficient, special auxiliary colorectal cancer, can be used as an index that judges patient's prognosis, and to the detection of colorectal cancer, fundamental research and detection provide reference, have Clinical practicability and scientific research.
A kind of immunohistochemical staining for colorectal cancer detects the application of (looking into) kit in investigation of clinical epidemiology, and this kit is comprised of following material:
A) antibody of the anti-human Interleukin-17F (IL-17F) in rabbit source.
B) biotin labeled goat anti-rabbit igg;
C) HRP of Avidin mark;
D) normal goats serum confining liquid;
E) 3% (volume ratio) H
2o
2;
F) DAB nitrite ion;
G) haematoxylin;
It checks that step is:
1. the tissue paraffin embedding, cut into slices, and paraffin section was placed in 58-62 ℃ of baking box after 1 hour, dewaxes to water, with PBS (pH7.4), rinses 2-4 time, each 4-6 minute.
2. antigen Microwave method: 10mM pH 5.8-6.2 sodium citrate buffer is poured in the Microwave method box and section is put into and repair liquid, the high fiery 3min of micro-wave oven → moderate heat 7min → low fiery 3min.Naturally after cooling, to room temperature (20-25 ℃), PBS rinses 2-4 time, each 4-6 minute.
3. 3% (volume ratio) H is put in section
2o
2solution, hatch under room temperature 15 minutes, with the blocking-up endogenous peroxydase.PBS rinses 2-4 time, each 4-6 minute.
4. get rid of PBS liquid, drip 5%BSA room temperature 28-32 minute.
5. get rid of BSA, first antibody is pressed the concentration dilution of 1: 100.Every section adds 100 μ l dilutions to cover tissue, and 4 ℃ are spent the night.PBS rinses 2-4 time, each 4-6 minute.
6. get rid of PBS liquid, every section adds 100 μ l second antibody, under room temperature, hatches 45 minutes.PBS rinses 2-4 time, each 4-6 minute.
7. get rid of PBS liquid, every section adds the freshly prepared DAB solution of 50-100 μ l, under room temperature (20-25 ℃, identical up and down), hatches 4-6 minute, and microscope is controlled colour developing (about 1min).
8. after colour developing fully, distilled water or tap water rinse, and haematoxylin is redyed 1-3min, from the beginning washing.
9. section dehydrates through gradient alcohol (70-100%), and dimethylbenzene is transparent, the neutral gum sealing.The observation of result:
IL-17F protein positive product mainly is positioned cytoplasm, shows as in endochylema brown yellow granule is arranged.Express in the normal bowel epithelial cell, and do not express in the enterocyte of canceration or low the expression.The immunohistochemical staining result adopts following criterion.Dye levels is marked: colourless note 0 minute, faint yellow note 1 minute, brown color note 2 minutes and sepia are remembered 3 minutes; The shared number percent scoring by positive cell again: 0 minute negative, positive cell number≤10% note 1 minute, 11%～50% note 2 minutes, 51%～75% note 3 minutes,>75% note 4 minutes, then calculate both product.Product<3 are minute negative, and >=3 minutes positive.
The present invention compared with prior art has the following advantages and effect:
This antibody test has high sensitivity, and the characteristics of high specific and high accuracy not only can be distinguished cancerous issue and normal structure, can also distinguish cell and the normal cell of canceration in cancerous issue.The IL-17F dyeing that is positive in normal colonic tissue's epithelial cell, and be negative in the cancerous tumor cell epithelium.
The applicant has analyzed the expression of IL-17F in Patients with Colorectal Cancer excision sample by Real-time quantitative PCR and western blotting technique.Find in 20 routine patient's specimens from pri, the mRNA level of cancerous issue IL-17F is significantly lower than normal structure (p<0.05).In 4 routine patient's specimens from pri, the protein level of cancerous issue IL-17F is significantly lower than normal structure (p<0.05).
The accompanying drawing explanation
Fig. 1 is a kind of immunohistochemical staining figure;
The colored graph that AC is normal colon (be the end of patient's excision sample, away from the normal structure of focal zone).The colored graph that BD is canceration focal zone colon (being the centre of patient's excision sample, the cancerous issue of focus center).AB is that histotomy carries out immunohistochemical staining figure with IL-17F antibody, and CD is that blank dyeing is that histotomy carries out immunohistochemical staining, does not add IL-17F antibody.In 4 figure, the only dyeing (arrow indication) that is positive of the epithelial cell in A figure, the cell dyeing that all is negative in remaining figure;
Fig. 2 is a kind of immunohistochemical staining figure;
The cancerous issue immunohistochemical staining figure that Fig. 2 is focus zone in a kind of patient's excision sample.In figure, the A position is to change and observe relatively normal colonic epithelium from nucleus, and the B position is for changing and observe the colonic epithelium that canceration has occurred from nucleus.The dyeing that is positive of the cell of A position, the dyeing that is negative of the cell of B position.
2 figure results show the epithelial cell high expressed IL-17F of normal colonic tissue and the cancerous issue epithelial cell low/substantially do not express IL-17F.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Be to be understood that, these embodiment only are not used in restriction the scope of protection of present invention for the present invention is described, not marked concrete experiment condition and method in the following example, usually according to normal condition as fine works molecular biology guide, F.M. the chief editor such as Ao Sibai, Science Press, 1995, molecular cloning experiment guide (third edition); D.L. Spector etc., Science Press, 2001, cell experiment guide; Lv Hongsheng, Science Press, 1982, or connect the condition of advising according to manufacturer.
Embodiment 1:
A kind of kit of the immunohistochemical staining for detection of colorectal cancer, this immunohistochemical staining kit comprises:
A) antibody of the anti-human Interleukin-17F (IL-17F) in rabbit source;
B) biotin labeled goat anti-rabbit igg;
C) HRP of Avidin mark;
D) normal goats serum confining liquid;
E) 3% (volume ratio) H
2o
2;
F) DAB nitrite ion;
G) haematoxylin;
B-g is Wuhan Boster Biological Technology Co., Ltd.'s product.
Key is the antibody preparation of the anti-human Interleukin-17F (IL-17F) in rabbit source in the present invention, and its method comprises the following steps:
A) build the IL-17F expression vector.Kit
-Inducible/Secreted Kit with pCoHygro is purchased from invitrogen company.The Fc fragment label of people's Immunoglobulin IgG1 is cloned in kit in the pMT/BiP/V5-HisA expression plasmid, obtains fruit bat expression system-immunoglobulin (Ig) fusion plasmid (DES-Ig).The cDNA sequence of method amplification human il-17 F by PCR, be cloned into the DES-Ig plasmid.
B) drosophila cell of setting up stable transfection IL-17F expression vector is S2 (cell provided in kit), copper sulphate inducible protein secreting, expressing purifying IL-17F.According to the kit instructions (
-Inducible/Secreted Kit with pCoHygro kit), by the pCoHygro plasmid co-transfection drosophila cell provided in the above-mentioned carrier successfully constructed and kit, be S2, screening obtains stable cell lines; And with the secreting, expressing of copper sulphate inducible protein.Collect supernatant, use albumin A-agarose (Protein A-Sepharose) pillar purchased from the enriching and purifying IL-17F-Ig of sigma company fusion.
C) immune rabbit obtains the polyclonal antibody of IL-17F and identifies.The conventional method immune rabbit, collect serum.Concrete steps are that to get the 200ug immunogene be that IL-17F-Ig fusion and Freund's complete adjuvant carry out being mixed to form emulsion at 1: 1, are injected into the subcutaneous and rear leg muscle of rabbit both shoulders; The 2 weeks booster immunizations in interval, immune commercial weight reduces by half, and is mixed into the emulsion injection with incomplete Freunds adjuvant and closes on position; 2 weeks booster immunizations in interval again; Within the 7th day, get blood after injection.Draw serum, centrifugal, packing, frozen.Detect antibody titer.
Utilize the specific polyclonal antibody of anti-IL-17F in the present invention, conventional reagent (the biotin labeled goat anti-rabbit igg of combined immunization histochemical stain; The HRP of Avidin mark; Normal goats serum confining liquid; 3%H
2o
2; The DAB nitrite ion; Haematoxylin), the pathology detection of convenient, efficient, special auxiliary colorectal cancer, can be used as an index that judges patient's prognosis, and to the detection of colorectal cancer, fundamental research and detection provide reference, have Clinical practicability and scientific research.
The application of a kind of kit of the immunohistochemical staining for detection of colorectal cancer in investigation of clinical epidemiology, this kit is comprised of following material:
A) antibody of the anti-human Interleukin-17F (IL-17F) in rabbit source.
B) biotin labeled goat anti-rabbit igg;
C) HRP of Avidin mark;
D) normal goats serum confining liquid;
E) H of 3% (volume ratio)
2o
2;
F) DAB nitrite ion;
G) haematoxylin;
Its detecting step is:
1. the tissue paraffin embedding, cut into slices, and paraffin section was placed in 60 ℃ of baking boxs after 1 hour, dewaxed to water, rinsed three times each 5 minutes with PBS (pH7.4).
2. antigen Microwave method: 10mM pH 6.0 ± 0.1 sodium citrate buffers are poured in the Microwave method box and section are put into and repair liquid, the high fiery 3min of micro-wave oven → moderate heat 7min → low fiery 3min.After naturally cooling to room temperature (20-25 ℃), PBS rinses three times, each 5 minutes.
3. 3% (volume ratio) superoxol is put in section, under room temperature, hatches 15 minutes, with the blocking-up endogenous peroxydase.PBS rinses three times, each 5 minutes.
4. get rid of PBS liquid, drip 5%BSA room temperature 30 minutes.
5. get rid of BSA, first antibody is pressed the concentration dilution of 1: 100.Every section adds 100 μ l dilutions to cover tissue, and 4 ℃ are spent the night.PBS rinses three times, each 5 minutes.
6. get rid of PBS liquid, every section adds 100 μ l second antibody, under room temperature, hatches 45 minutes.PBS rinses three times, each 5 minutes.
7. get rid of PBS liquid, every section adds the freshly prepared DAB solution of 50-100 μ l, under room temperature (20-25 ℃, identical up and down), hatches 5 minutes, and microscope is controlled colour developing (about 1min).
8. after colour developing fully, distilled water or tap water rinse, and haematoxylin is redyed 2min, from the beginning washing.
9. section dehydrates through gradient alcohol (70-100%), and dimethylbenzene is transparent, the neutral gum sealing.The observation of result:
IL-17F protein positive product mainly is positioned cytoplasm, shows as in endochylema brown yellow granule is arranged.Express in the normal bowel epithelial cell, and do not express in the enterocyte of canceration or low the expression.The immunohistochemical staining result adopts following criterion.Dye levels is marked: colourless note 0 minute, faint yellow note 1 minute, brown color note 2 minutes and sepia are remembered 3 minutes; The shared number percent scoring by positive cell again: 0 minute negative, positive cell number≤10% note 1 minute, 11%～50% note 2 minutes, 51%～75% note 3 minutes,>75% note 4 minutes, then calculate both product.Product<3 are minute negative, and >=3 minutes positive.
Claims (1)
1. the immunohistochemical staining detection kit of a colorectal cancer, this kit contains:
A) antibody of the anti-human Interleukin-17F in rabbit source;
B) biotin labeled goat anti-rabbit igg;
C) HRP of Avidin mark;
D) normal goats serum confining liquid;
E) 3% volume ratio H
2o
2;
F) DAB nitrite ion;
G) haematoxylin;
The preparation method for antibody of the anti-human Interleukin-17F in described rabbit source is as follows:
A) build the IL-17F expression vector, the Fc fragment label of people's Immunoglobulin IgG1 is cloned in kit in the pMT/BiP/V5-HisA expression plasmid, obtain fruit bat expression system-immunoglobulin (Ig) fusion plasmid DES-Ig, the cDNA sequence of method amplification human il-17 F by PCR, be cloned into the DES-Ig plasmid;
B) drosophila cell of setting up stable transfection IL-17F expression vector is S2, copper sulphate inducible protein secreting, expressing purifying IL-17F, by the pCoHygro plasmid co-transfection drosophila cell provided in the carrier that successfully constructs and kit, be S2, screening obtains stable cell lines; And, with the secreting, expressing of copper sulphate inducible protein, collect supernatant, with albumin A-agarose pillar enriching and purifying IL-17F-Ig fusion;
C) immune rabbit obtains the polyclonal antibody of IL-17F and identifies, the conventional method immune rabbit, collect serum, get the 200ug immunogene and be IL-17F-Ig fusion and Freund's complete adjuvant and carry out 1:1 and be mixed to form emulsion, be injected into the subcutaneous and rear leg muscle of rabbit both shoulders; The 2 weeks booster immunizations in interval, immune commercial weight reduces by half, and with incomplete Freunds adjuvant, is mixed into the emulsion injection; 2 weeks booster immunizations in interval again; Within the 7th day, get blood after injection, draw serum, centrifugal, packing, frozen.
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