CN105044348A - SALL4 immunohistochemical detection kit for diagnosis of lung cancer - Google Patents
SALL4 immunohistochemical detection kit for diagnosis of lung cancer Download PDFInfo
- Publication number
- CN105044348A CN105044348A CN201510069543.3A CN201510069543A CN105044348A CN 105044348 A CN105044348 A CN 105044348A CN 201510069543 A CN201510069543 A CN 201510069543A CN 105044348 A CN105044348 A CN 105044348A
- Authority
- CN
- China
- Prior art keywords
- agent
- minutes
- section
- sall4
- gained
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses an SALL4 immunohistochemical detection kit for diagnosis of lung cancer, a use method and an application of the kit and belongs to the field of medical and biological detection. The kit includes the following parts: positive control, negative control, a primary antibody, a secondary antibody, a fixing agent, a defatting and clarifying agent, a rehydration agent, a dehydration agent, a buffer solution A, a buffer solution B, an antigen repairing agent, a permeating agent, a peroxidase inactivating agent, a non-specific protein blocking agent, a color developing solution and a coloring agent. The kit is high in specificity and sensitivity and allows differential expression, not only can effectively distinguish lung cancer tissue from normal tissue but also can provides new evidence for diagnosis and classification to lung cancer individualized treatment, so that detection time of lung cancer can be advanced as more as possible, thereby solving the problem that the detection means in the prior art are low in sensitivity and are difficult to achieve early-stage detection of lung cancer. The kit has high clinically applying value and social benefit.
Description
Technical field
The present invention relates to a kind of SALL4 Immunohistochemical detection kit for pulmonary cancer diagnosis, using method and application, belong to medical science and Biological Detection field.
Background technology
Lung cancer is one of current global modal malignant tumour, and endangering serious and in rising trend, is the disease that global incidence and mortality ratio increase are the fastest.The high case fatality rate of lung cancer is mainly owing to accomplishing early diagnosis, and therefore, the early diagnosis of lung cancer is timely Canonical management and improves the most effective measures of its prognosis.Diagnostic method traditional at present depends on iconography and fiberoptic bronchoscopy, but these two kinds of methods have complicated operation, have radiation spinoff and the high limitation of expense.Discovery and the rationally application of tumor markers are significant to tumour early detection, early diagnosis.
Tumor markers is that tumour cell generates due to the change of the expression of gene or the antigen that reduces and other bioactivator in Carcinogenesis, can be used for the early diagnosis of tumour, by stages, monitoring tumor progression and evaluate the result for the treatment of of medicine.Tumor markers can bring tremendous influence to the clinical treatment of tumour, especially when it can be detected before clinical disease occurs, or when may be used for the real-time detection of result for the treatment of.Compared with traditional diagnostic means, tumor markers detect have easy, expense is relatively low, do not have the advantages such as radiation hazard, risk are little, patient is acceptant.At present, in order to the demand of the clinical diagnosis and treatment that meet tumour, the research and development of tumor markers are urgently accelerated.
At present for the tumor markers of early diagnosis of tumor, mostly can not widespread use in reality detects due to shortage sensitivity and specificity.Such as, for liver cancer, alpha-fetoprotein and ultrasonic inspection are the modes of the diagnosis high risk patient generally adopted, and really significantly improve the survival rate of hepatocarcinoma patient, but remolding sensitivity is lower; Tumour antigen CA-125 has higher sensitivity, but lacks specificity.Similarly, for breast cancer detection neoplastic hematologic disorder mark CA15-3 because of sensitivity low, in early days diagnosis in almost useless.Early diagnosis and the differentiation that is optimum and malignant tumour of tumour remain a clinical problem, need new techniques and methods to find the sensitivity that new tumor markers and raising tumor markers detect and confidence level.And for the lung cancer of high incidence and mortality ratio, find hypersensitivity and specific mark, the Diagnosis and Treat of lung cancer is significant, become the task of top priority of lung cancer control.
SALL4 albumen is encoded by human source gene SALL4 and is translated, its full name is Sal sample albumen 4 (Sal-likeprotein4), another name zinc finger protein 797 (Zincfingerprotein797) and zinc finger protein SALL4 (ZincfingerproteinSALL4).SALL4 belongs to SalC2H2 putative zinc finger protein family, includes C2H2 class zinc fingers.SALL4 albumen has two hypotypes, i.e. SALL4A and SALL4B, and its molecular size range is respectively 112.2KD and 65.7KD.SALL4 albumen is mainly distributed in tenuigenin and nucleus.
SALL4 belongs to zinc finger transcription factor, by interacting with OCT3/4, SOX2 and NANOG, participates in self and the maintenance of stem cell.SALL4 is high expressed in embryonic stem cell not only, and in human hematopoietic system tumour also high expressed, such as acute myelogenous and lymphocytic leukemia.Further discovery SALL4 can raise the expression of proto-oncogene Bmi-1 in human hematopoietic stem cell and leukaemia.Some other research shows, SALL4 can as tumor marker, in the diagnosis of lung cancer and breast cancer, have higher using value.SALL4mRNA is respectively 85.1% and 92.9% at lung cancer Sensitivity and Specificity, and the lung cancer specimen SALL4mRNA of 93% is the twice on cancer side.About the expression of SALL4 albumen in lung carcinoma cell, and carry out the feasibility of pulmonary cancer diagnosis as a kind of lung cancer marker, there is no relevant report at present.
At present, clinically, immunohistochemical staining is widely applied in pathologic diagnosis of tumor, relates to field and comprises the discriminating of tumor tissues origin, the judgement not breaking up malignant tumour character, the discriminating of various morphological system tumour, determines tumour original site etc.Its principle be with mark antibody on cell or in-house corresponding antigen carry out qualitative, location or quantitatively detect, present eye-catching positive color through histochemical color reaction, then use optical microscope, fluorescent microscope or electron microscope observation.
Summary of the invention
An object of the present invention, is to provide a kind of SALL4 Immunohistochemical detection kit for pulmonary cancer diagnosis and using method thereof, can effectively distinguishes cancerous lung tissue and normal structure, also can be lung cancer individualized treatment and provide new diagnosis and classification foundation.
Object two of the present invention, is to provide a kind of application of the SALL4 Immunohistochemical detection kit for pulmonary cancer diagnosis.
The technical scheme that the present invention solves the problems of the technologies described above is as follows: a kind of SALL4 Immunohistochemical detection kit for pulmonary cancer diagnosis, comprises following component: positive control, negative control, primary antibodie, two anti-, fixing agent, degreasing and clarifier, rehydration agent, dewatering agent, buffer A, buffer B, antigen retrieval agent, penetrating dose, peroxidase deactivator, nonspecific proteins blocking agent, nitrite ion and coloring agent.
On the basis of technique scheme, the present invention can also do following improvement.
Further, described positive control is the spermatogonium tumor tissue of SALL4 high expressed, and described negative control is: not containing the lung squamous cell carcinoma cancers that the sheep blood serum of SALL4 antibody is hatched.
Further, described primary antibodie is the anti-human SALL4 monoclonal antibody in mouse source, described two resist for biotin labeled goat anti-mouse IgG, described fixing agent is formalin, described degreasing and clarifier are dimethylbenzene, described rehydration agent is concentration is 100%v/v, 90%v/v, 80%v/v, the graded ethanol of 70%v/v, described dewatering agent is concentration is 70%v/v, 80%v/v, 90%v/v, the graded ethanol of 100%v/v, described buffer A is 1mMPBS (PH7.4), described buffer B is 1mMPBST (PH7.4), described antigen retrieval agent is 1mMTris/EDTA (PH8), described penetrating dose is 0.3%Triton-100, described peroxidase deactivator is the hydrogen peroxide of 3%v/v, described nonspecific proteins blocking agent is 0.5% sheep blood serum, described nitrite ion is DAB, described coloring agent is haematine.
For a using method for the SALL4 Immunohistochemical detection kit of pulmonary cancer diagnosis, comprise the steps:
(1) get testing sample and positive control to carry out formalin respectively and fix, place 12 hours for 4 DEG C;
(2) by step gained (1) fixed preparation, carry out paraffin embedding, make 4 μm of thick paraffin sections, be placed in 37 DEG C of dryings 12 hours, obtain dried section;
(3) dewax rehydration: by the dried section of step (2) gained, in 60 DEG C of bakings 2 hours, then dewaxes 5 minutes with degreasing and clarifier, then use rehydration agent rehydration, every gradient 2 minutes, then use aseptic water washing 2 minutes, obtains rehydration section;
(4) step (3) gained rehydration cut into slices, add penetrating dose, carry out penetrating to tissue, room temperature places 3 minutes, removes penetrating dose, then rinses 5 minutes with washing agent, obtains penetrating section;
(5) by the penetrating section of step (4) gained, soak 10 minutes with peroxidase deactivator, carry out endogenous peroxidase activity and close, then rinse 5 minutes by buffer A, obtain the section of intrinsic oversxidase inactivation;
(6) cut into slices by step (5) gained endogenous peroxydase inactivation, be placed in antigen retrieval agent, boiling water boiling 3 minutes, carries out antigen retrieval, and room temperature cools, and rinses 5 minutes by buffer A, obtains antigen retrieval section;
(7) step (6) gained antigen retrieval cut into slices, add nonspecific proteins blocking agent, normal temperature closes 1 hour, obtains non-specific antibody and closes section;
(8) step (7) gained non-specific antibody is closed section, except negative control only adds nonspecific proteins blocking agent, all the other sections all add the primary antibodie of 10 μ g/ml of nonspecific proteins blocking agent dilution, then incubated at room 2 hours, rinse by buffer A and buffer B successively, each flushing 3 times, each 5 minutes, obtains the section after primary antibodie;
(9) by the section after step (8) gained primary antibodie, add 200 times of nonspecific proteins blocking agent dilutions two resist, incubated at room 1 hour, rinse by buffer A and buffer B successively, each flushing 3 times, each 5 minutes, obtain two anti-after section;
(10) cut into slices after anti-for step (9) gained two, dye with freshly prepared nitrite ion, incubated at room 5 minutes, obtain the section after dyeing;
(11) section after step (10) gained being dyeed, tap water 3 minutes, coloring agent redyes 3 minutes, and tap water 3 minutes, obtains redying section;
(12) step (11) gained is redyed section, dehydrate with dewatering agent, every gradient 2 minutes, then use degreasing and transparent 5 minutes of clarifier, neutral gum sealing, obtain the section of SALL4 immunohistochemical staining;
(13) step (12) gained SALL4 immunohistochemical staining is cut into slices, under being placed in optical microscope, observe dye levels, statistics positive cell number, then in conjunction with the result of positive control and negative control, judge whether testing sample is cancerous lung tissue.
On the basis of technique scheme, the present invention can also do following improvement.
Further, step (1) described testing sample is the tissue samples that takes out from patient lung tissue or cell mass.
Further, the described freshly prepared nitrite ion of step (10) is with distilled water: DAB substrate storage liquid: stable superoxide: hyperchromic liquor capacity proportioning is that 17:1:1:1 is formulated.
Further, the enlargement factor of step (13) described optical microscope is 400 times.
Employing is widely used in immunohistochemistry positive reaction classification, the Tanaka that accuracy is high improves quantitative score method and judges whether testing sample is cancerous lung tissue, concrete judgment criteria is: every routine random observation 5 visuals field, each visual field counts 200 cells, counting cells sum and positive cell number, by the percentage score shared by positive cell, obtain numerical value a: positive cell rate≤5%, be designated as 1 point, 5%< positive cell rate≤25%, be designated as 2 points, 25%< positive cell rate≤50%, be designated as 3 points, 50%< positive cell rate, be designated as 4 points, meanwhile, staining power is scored, obtains numerical value b: be colourless, be designated as 0 point, faint yellow, be designated as 1 point, brown color, be designated as 2 points, sepia, be designated as 3 points, above-mentioned numerical value a, b are multiplied, obtain numerical value c, after again the numerical value c in 5 visuals field being added, average, above-mentioned mean value is divided into 4 grades: 0 point≤mean value≤1 point, be negative (-), 2 points≤mean value≤4 point is the weak positive (+), 5 points≤mean value≤8 point, be moderate positive (++), 9 points≤mean value≤12 point, be strong positive (+++).
For an application for the SALL4 Immunohistochemical detection kit of pulmonary cancer diagnosis, the lung cancer of described kit diagnosis is small-cell carcinoma of the lung and non-small cell lung cancer.
In the present invention, " normal tissue cell " refers to the cell in the activity of biosome or tissue with normal function.Molecular labeling of the present invention is preferably at the common detected molecular labeling of the tumour cell of multiple cancer kind, can use as single mark in multiple cancer kind, but this molecule is marked at different cancer species diversity and expresses, can as a foundation of cancer kind classification.Molecular labeling of the present invention can't detect at the cancer parietal cell of non-tumor cell.In this instructions, " expression " refers to and can confirm expression product by methods known to those skilled in the art such as such as RT-PCR, in situ hybridization, immunohistochemistry, chromatographys.In addition, " can't detect " and refer to cannot confirm expression product in the confirmation method of aforementioned expression product.In the present invention, judge that object is preferably human tissue and/or derives from the cell of people, but also can be the animal beyond people and the tissue deriving from this animal.In the present invention, " Tanaka improve quantitative score method " refer to by Sinicrope1995 first Application, by the quantitative point-score of Tanaka in improvement in 2000, this quantitative point-score is used for immunohistochemistry results positive grading evaluation, especially cancer detection, and accuracy is high, be widely used.
The invention has the beneficial effects as follows:
1. kit of the present invention, has that specificity is high, susceptibility is high, the feature of differential expression, not only can effectively distinguish cancerous lung tissue and normal structure, also can be lung cancer individualized treatment and provide new diagnosis and classification foundation.
2. kit of the present invention can improve the accuracy that lung cancer detects, and solves the problem of a lot of existing detection means poor accuracy.
3. the present invention can by the detection time of lung cancer in advance farthest, improve the survival rate of patients with lung cancer, for the control of lung cancer clinically provides important scientific basis, solve the problem that existing detection means sensitivity is all relatively low, be difficult to realize the early monitoring of lung cancer, there is higher clinical value and social benefit.
Accompanying drawing explanation
Fig. 1 is the application of SALL4 Immunohistochemical detection kit of the present invention in lung cancer detection.
In figure, (A) in normal lung tissue's negative control negative (-), (B) the weak positive (+) in adenocarcinoma of lung, (C) strong positive (+++) in small-cell carcinoma of the lung, (D) be strong positive (+++) in squamous cell lung carcinoma.
Embodiment
Be described principle of the present invention and feature below in conjunction with accompanying drawing, example, only for explaining the present invention, is not intended to limit scope of the present invention.
For a SALL4 Immunohistochemical detection kit for pulmonary cancer diagnosis, comprise following component: positive control, negative control, primary antibodie, two anti-, fixing agent, degreasing and clarifier, rehydration agent, dewatering agent, buffer A, buffer B, antigen retrieval agent, penetrating dose, peroxidase deactivator, nonspecific proteins blocking agent, nitrite ion and coloring agent.
Described positive control is SALL4 high expressed tissue, and described negative control is do not hatch tissue containing the sheep blood serum of SALL4 antibody.
Described primary antibodie is the anti-human SALL4 monoclonal antibody in mouse source, described two resist for biotin labeled goat anti-mouse IgG, described fixing agent is formalin, described degreasing and clarifier are dimethylbenzene, described rehydration agent is concentration is 100%v/v, 90%v/v, 80%v/v, the graded ethanol of 70%v/v, described dewatering agent is concentration is 70%v/v, 80%v/v, 90%v/v, the graded ethanol of 100%v/v, described buffer A is 1mMPBS (PH7.4), described buffer B is 1mMPBST (PH7.4), described antigen retrieval agent is 1mMTris/EDTA (PH8), described penetrating dose is 0.3%Triton-100, described peroxidase deactivator is the hydrogen peroxide of 3%v/v, described nonspecific proteins blocking agent is 0.5% sheep blood serum, described nitrite ion is DAB, described coloring agent is haematine.
For a using method for the SALL4 Immunohistochemical detection kit of pulmonary cancer diagnosis, comprise the steps:
(1) get 135 routine cancerous lung tissues, 5 routine normal structures and spermatogonium tumor tissue, carry out formalin respectively and fix, place 12 hours for 4 DEG C, wherein said cancerous lung tissue is the tissue samples taken out from patient lung tissue;
(2) by step gained (1) fixed preparation, carry out paraffin embedding, make 4 μm of thick paraffin sections, be placed in 37 DEG C of dryings 12 hours, obtain dried section;
(3) dewax rehydration: by the dried section of step (2) gained, in 60 DEG C of bakings 2 hours, then dewaxes 5 minutes with degreasing and clarifier, then use rehydration agent rehydration, every gradient 2 minutes, then use aseptic water washing 2 minutes, obtains rehydration section;
(4) step (3) gained rehydration cut into slices, add penetrating dose, carry out penetrating to tissue, room temperature places 3 minutes, removes penetrating dose, then rinses 5 minutes with washing agent, obtains penetrating section;
(5) by the penetrating section of step (4) gained, soak 10 minutes with peroxidase deactivator, carry out endogenous peroxidase activity and close, then rinse 5 minutes by buffer A, obtain the section of intrinsic oversxidase inactivation;
(6) cut into slices by step (5) gained endogenous peroxydase inactivation, be placed in antigen retrieval agent, boiling water boiling 3 minutes, carries out antigen retrieval, and room temperature cools, and rinses 5 minutes by buffer A, obtains antigen retrieval section;
(7) step (6) gained antigen retrieval cut into slices, add nonspecific proteins blocking agent, normal temperature closes 1 hour, obtains non-specific antibody and closes section;
(8) step (7) gained non-specific antibody is closed section, except negative control only adds nonspecific proteins blocking agent, all the other sections all add the primary antibodie of 10 μ g/ml of nonspecific proteins blocking agent dilution, then incubated at room 2 hours, rinse by buffer A and buffer B successively, each flushing 3 times, each 5 minutes, obtains the section after primary antibodie;
(9) by the section after step (8) gained primary antibodie, add 200 times of nonspecific proteins blocking agent dilutions two resist, incubated at room 1 hour, rinse by buffer A and buffer B successively, each flushing 3 times, each 5 minutes, obtain two anti-after section;
(10) cut into slices after anti-for step (9) gained two, dye with freshly prepared nitrite ion, incubated at room 5 minutes, obtain the section after dyeing, wherein said freshly prepared nitrite ion is with distilled water: DAB substrate storage liquid: stable superoxide: hyperchromic liquor capacity proportioning is that 17:1:1:1 is formulated;
(11) section after step (10) gained being dyeed, tap water 3 minutes, coloring agent redyes 3 minutes, and tap water 3 minutes, obtains redying section;
(12) step (11) gained is redyed section, dehydrate with dewatering agent, every gradient 2 minutes, then use degreasing and transparent 5 minutes of clarifier, neutral gum sealing, obtain the section of SALL4 immunohistochemical staining;
(13) step (12) gained SALL4 immunohistochemical staining is cut into slices, being placed in enlargement factor is under the optical microscope of 400 times, observe dye levels, statistics positive cell number, again in conjunction with the result of positive control and negative control, judge whether testing sample is cancerous lung tissue.
Adopt Tanaka to improve quantitative score method and judge whether testing sample is cancerous lung tissue, concrete judgment criteria is: every routine random observation 5 visuals field, each visual field counts 200 cells, counting cells sum and positive cell number, by the percentage score shared by positive cell, obtain numerical value a: positive cell rate≤5%, be designated as 1 point, 5%< positive cell rate≤25%, be designated as 2 points, 25%< positive cell rate≤50%, be designated as 3 points, 50%< positive cell rate, is designated as 4 points; Meanwhile, staining power is scored, obtains numerical value b: be colourless, be designated as 0 point, faint yellow, be designated as 1 point, brown color, be designated as 2 points, sepia, be designated as 3 points; Above-mentioned numerical value a, b are multiplied, obtain numerical value c, after again the numerical value c in 5 visuals field being added, average, above-mentioned mean value is divided into 4 grades: 0 point≤mean value≤1 point, be negative (-), 2 points≤mean value≤4 point is the weak positive (+), 5 points≤mean value≤8 point, be moderate positive (++), 9 points≤mean value≤12 point, be strong positive (+++).
Interpretation of result: find that SALL4 albumen is mainly expressed in cytoplasm, part is positioned at cell membrane.The positives dyeing of cytoplasm shows as yellow particle, and background is not painted.Strong positive (+++) in spermatogonium tumor tissue, in normal lung tissue negative (-), differential expression in cancerous lung tissue, be embodied in: the weak positive (+) in adenocarcinoma of lung, strong positive (+++) in small cell carcinoma, strong positive (+++) in lung squamous cancer.
As shown in Figure 1, the application of SALL4 Immunohistochemical detection kit in lung cancer detection, wherein, (A) in normal lung tissue's negative control negative (-), (B) the weak positive (+) in adenocarcinoma of lung, (C) strong positive (+++) in small-cell carcinoma of the lung, (D) be strong positive (+++) in squamous cell lung carcinoma.
Table 1 cancer sample multi-class classification
| Lung Cancer Types | Number of cases |
| Squamous cell carcinoma | 64 |
| Gland cancer | 35 |
| Papillary adenocarcinoma | 9 |
| Adenosquamous carcinoma | 9 |
| Small cell carcinoma | 7 |
| Pulmonary artery neoplasms | 7 |
| Metastatic carcinoma | 2 |
| Carcinoid tumor | 1 |
| Undifferentiated cancer | 1 |
| Amount to | 135 |
After testing, in 135 routine lung cancer samples, SALL4 is positive in 119 examples, as shown in table 2.
Table 2SALL4 Immunohistochemical detection kit lung cancer positive rate is analyzed
| Mark | Normal and non-lung cancer organizes positive rate (%) | Cancerous lung tissue positive rate (%) | Chi-square Test X2/P |
| SALL4 | O％(0/10) | 88％(119/135) | 3.53/0.003 |
SALL4 Immunohistochemical detection kit detection of lung cancer Clinical symptoms correlation analysis
Select age, sex, lymphatic metastasis, cancer kind to be parameter, use Chi-square Test to analyze above-mentioned 135 routine cancer samples, to verify lung cancer marker SALL4 and whether to have correlativity with lung cancer clinical feature.
Through inspection, the expression of discovery SALL4 and patient age, sex, lymphatic metastasis, Cancer pathologies type and lung cancer do not have the correlativity of conspicuousness period, as shown in table 3.
Table 3SALL4 Immunohistochemical detection kit detection of lung cancer Clinical symptoms correlation analysis
SALL4 Immunohistochemical detection kit lung cancer Sensitivity and Specificity is analyzed
By the expression of immunohistochemical analysis SALL4 in cancerous lung tissue and normal structure, judge the Sensitivity and Specificity of this kit, and by observing its differential expression, judge cancer species.Analyze above-mentioned 135 routine cancer samples, find that SALL4 is positive in 119 routine cancerous lung tissues, and be negative in non-lung cancer tissue, its Sensitivity and Specificity reaches 88% and 100% respectively, as shown in table 4.And find further, SALL4 is variant in gland cancer, squama cancer and the positives expression of small cell carcinoma, wherein, SALL4 gland cancer and squama cancer positives comparatively strong, express in small-cell carcinoma of the lung and relatively weaken.Show thus, SALL4 Immunohistochemical detection kit can as a very useful pulmonary cancer diagnosis mark kit.
Table 4SALL4 Immunohistochemical detection kit lung cancer Sensitivity and Specificity is analyzed
| Mark | Susceptibility (cancerous lung tissue, positive) | Specificity (non-lung cancer tissue, negative) |
| SALL4 | 88％(119/135) | 100％(10/10) |
For an application for the SALL4 Immunohistochemical detection kit of pulmonary cancer diagnosis, the lung cancer of described kit diagnosis is small-cell carcinoma of the lung and non-small cell lung cancer.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. the SALL4 Immunohistochemical detection kit for pulmonary cancer diagnosis, it is characterized in that, comprise following component: positive control, negative control, primary antibodie, two anti-, fixing agent, degreasing and clarifier, rehydration agent, dewatering agent, buffer A, buffer B, antigen retrieval agent, penetrating dose, peroxidase deactivator, nonspecific proteins blocking agent, nitrite ion and coloring agent.
2. a kind of SALL4 Immunohistochemical detection kit for pulmonary cancer diagnosis according to claim 1, it is characterized in that, described positive control is the spermatogonium tumor tissue of SALL4 high expressed, and described negative control is not containing the lung squamous cell carcinoma cancers that the sheep blood serum of SALL4 antibody is hatched.
3. a kind of SALL4 Immunohistochemical detection kit for pulmonary cancer diagnosis according to claim 1, it is characterized in that, described primary antibodie is the anti-human SALL4 monoclonal antibody in mouse source, described two resist for biotin labeled goat anti-mouse IgG, described fixing agent is formalin, described degreasing and clarifier are dimethylbenzene, described rehydration agent is concentration is 100%v/v, 90%v/v, 80%v/v, the graded ethanol of 70%v/v, described dewatering agent is concentration is 70%v/v, 80%v/v, 90%v/v, the graded ethanol of 100%v/v, described buffer A is 1mMPBS (PH7.4), described buffer B is 1mMPBST (PH7.4), described antigen retrieval agent is 1mMTris/EDTA (PH8), described penetrating dose is 0.3%Triton-100, described peroxidase deactivator is the hydrogen peroxide of 3%v/v, described nonspecific proteins blocking agent is 0.5% sheep blood serum, described nitrite ion is DAB, described coloring agent is haematine.
4. as described in any one of claims 1 to 3 for a using method for the SALL4 Immunohistochemical detection kit of pulmonary cancer diagnosis, it is characterized in that, comprise the steps:
(1) get testing sample and positive control to carry out formalin respectively and fix, place 12 hours for 4 DEG C;
(2) by step gained (1) fixed preparation, carry out paraffin embedding, make 4 μm of thick paraffin sections, be placed in 37 DEG C of dryings 12 hours, obtain dried section;
(3) dewax rehydration: by the dried section of step (2) gained, in 60 DEG C of bakings 2 hours, then dewaxes 5 minutes with degreasing and clarifier, then use rehydration agent rehydration, every gradient 2 minutes, then use aseptic water washing 2 minutes, obtains rehydration section;
(4) step (3) gained rehydration cut into slices, add penetrating dose, carry out penetrating to tissue, room temperature places 3 minutes, removes penetrating dose, then rinses 5 minutes with washing agent, obtains penetrating section;
(5) by the penetrating section of step (4) gained, soak 10 minutes with peroxidase deactivator, carry out endogenous peroxidase activity and close, then rinse 5 minutes by buffer A, obtain the section of intrinsic oversxidase inactivation;
(6) cut into slices by step (5) gained endogenous peroxydase inactivation, be placed in antigen retrieval agent, boiling water boiling 3 minutes, carries out antigen retrieval, and room temperature cools, and rinses 5 minutes by buffer A, obtains antigen retrieval section;
(7) step (6) gained antigen retrieval cut into slices, add nonspecific proteins blocking agent, normal temperature closes 1 hour, obtains non-specific antibody and closes section;
(8) step (7) gained non-specific antibody is closed section, except negative control only adds nonspecific proteins blocking agent, all the other sections all add the primary antibodie of 10 μ g/ml of nonspecific proteins blocking agent dilution, then incubated at room 2 hours, rinse by buffer A and buffer B successively, each flushing 3 times, each 5 minutes, obtains the section after primary antibodie;
(9) by the section after step (8) gained primary antibodie, add 200 times of nonspecific proteins blocking agent dilutions two resist, incubated at room 1 hour, rinse by buffer A and buffer B successively, each flushing 3 times, each 5 minutes, obtain two anti-after section;
(10) cut into slices after anti-for step (9) gained two, dye with freshly prepared nitrite ion, incubated at room 5 minutes, obtain the section after dyeing;
(11) section after step (10) gained being dyeed, tap water 3 minutes, coloring agent redyes 3 minutes, and tap water 3 minutes, obtains redying section;
(12) step (11) gained is redyed section, dehydrate with dewatering agent, every gradient 2 minutes, then use degreasing and transparent 5 minutes of clarifier, neutral gum sealing, obtain the section of SALL4 immunohistochemical staining;
(13) step (12) gained SALL4 immunohistochemical staining is cut into slices, under being placed in optical microscope, observe dye levels, statistics positive cell number, then in conjunction with the result of positive control and negative control, judge whether testing sample is cancerous lung tissue.
5. using method according to claim 4, is characterized in that, step (1) described testing sample, is the tissue samples that takes out from patient lung tissue or cell mass.
6. using method according to claim 4, is characterized in that, the described freshly prepared nitrite ion of step (10) is with distilled water: DAB substrate storage liquid: stable superoxide: hyperchromic liquor capacity proportioning is that 17:1:1:1 is formulated.
7. using method according to claim 4, is characterized in that, the enlargement factor of step (13) described optical microscope is 400 times.
8. as described in any one of claims 1 to 3 for an application for the SALL4 Immunohistochemical detection kit of pulmonary cancer diagnosis, it is characterized in that, the lung cancer of described kit diagnosis is small-cell carcinoma of the lung and non-small cell lung cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510069543.3A CN105044348A (en) | 2015-02-10 | 2015-02-10 | SALL4 immunohistochemical detection kit for diagnosis of lung cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510069543.3A CN105044348A (en) | 2015-02-10 | 2015-02-10 | SALL4 immunohistochemical detection kit for diagnosis of lung cancer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105044348A true CN105044348A (en) | 2015-11-11 |
Family
ID=54451052
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510069543.3A Pending CN105044348A (en) | 2015-02-10 | 2015-02-10 | SALL4 immunohistochemical detection kit for diagnosis of lung cancer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105044348A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105628913A (en) * | 2016-01-13 | 2016-06-01 | 深圳中山生殖与遗传研究所 | Kit for evaluating endometrial receptivity and using method thereof |
| CN111766385A (en) * | 2020-07-06 | 2020-10-13 | 河南赛诺特生物技术有限公司 | Immunohistochemical kit for rapidly identifying lung cancer and sclerosing lung cell tumor in operation |
| CN111954816A (en) * | 2018-03-08 | 2020-11-17 | 英美偌科有限公司 | Method for detecting MAGEA4 |
| CN112534264A (en) * | 2018-05-03 | 2021-03-19 | 波尔图大学病理学和免疫学研究所(Ipatimup) | Microsatellite instability antigenic markers |
| CN117384291A (en) * | 2023-07-28 | 2024-01-12 | 武汉爱博泰克生物科技有限公司 | Anti-human SALL4 rabbit monoclonal antibody, preparation method and application thereof, polynucleotide molecule, expression vector and host cell |
| CN117384291B (en) * | 2023-07-28 | 2024-07-05 | 武汉爱博泰克生物科技有限公司 | Anti-human SALL4 rabbit monoclonal antibody, preparation method and application thereof, polynucleotide molecule, expression vector and host cell |
-
2015
- 2015-02-10 CN CN201510069543.3A patent/CN105044348A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105628913A (en) * | 2016-01-13 | 2016-06-01 | 深圳中山生殖与遗传研究所 | Kit for evaluating endometrial receptivity and using method thereof |
| CN111954816A (en) * | 2018-03-08 | 2020-11-17 | 英美偌科有限公司 | Method for detecting MAGEA4 |
| CN112534264A (en) * | 2018-05-03 | 2021-03-19 | 波尔图大学病理学和免疫学研究所(Ipatimup) | Microsatellite instability antigenic markers |
| CN111766385A (en) * | 2020-07-06 | 2020-10-13 | 河南赛诺特生物技术有限公司 | Immunohistochemical kit for rapidly identifying lung cancer and sclerosing lung cell tumor in operation |
| CN111766385B (en) * | 2020-07-06 | 2023-09-26 | 河南赛诺特生物技术有限公司 | Immunohistochemical kit for rapidly identifying lung cancer and sclerosing pulmonary cytoma in operation |
| CN117384291A (en) * | 2023-07-28 | 2024-01-12 | 武汉爱博泰克生物科技有限公司 | Anti-human SALL4 rabbit monoclonal antibody, preparation method and application thereof, polynucleotide molecule, expression vector and host cell |
| CN117384291B (en) * | 2023-07-28 | 2024-07-05 | 武汉爱博泰克生物科技有限公司 | Anti-human SALL4 rabbit monoclonal antibody, preparation method and application thereof, polynucleotide molecule, expression vector and host cell |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Asa | My approach to oncocytic tumours of the thyroid | |
| Jiang et al. | Alpha‐methylacyl‐CoA racemase: a multi‐institutional study of a new prostate cancer marker | |
| MAhAdevAPPA et al. | Diagnostic and prognostic significance of Ki-67 immunohistochemical expression in surface epithelial ovarian carcinoma | |
| Lin et al. | High-plex immunofluorescence imaging and traditional histology of the same tissue section for discovering image-based biomarkers | |
| CN106153922B (en) | A kind of colon cancer prognosis prediction mark and its detection method | |
| JP5370826B2 (en) | Cocktail antibody, discrimination kit and discrimination method for discriminating cancer tissue type | |
| Tannapfel et al. | Apoptosis and proliferation in relation to histopathological variables and prognosis in hepatocellular carcinoma | |
| Googe et al. | Preferentially expressed antigen in melanoma immunostaining in a series of melanocytic neoplasms | |
| CN105044348A (en) | SALL4 immunohistochemical detection kit for diagnosis of lung cancer | |
| Ramadan et al. | MicroRNA-21 expression, serum tumor markers, and immunohistochemistry in canine mammary tumors | |
| Zhang et al. | The role of SOX11 immunostaining in confirming the diagnosis of mantle cell lymphoma on fine‐needle aspiration samples | |
| Nikshoar et al. | Metas-Chip precisely identifies presence of micrometastasis in live biopsy samples by label free approach | |
| Ates Ozdemir et al. | PAX2, PAX8 and CDX2 expression in metastatic mucinous, primary ovarian mucinous and seromucinous tumors and review of the literature | |
| Matos et al. | Detection of lymph node micrometastases in malignant mammary tumours in dogs by cytokeratin immunostaining | |
| Zhu et al. | Six stroma-based RNA markers diagnostic for prostate cancer in European-Americans validated at the RNA and protein levels in patients in China | |
| Ohta et al. | HBME‐1 and CD15 immunocytochemistry in the follicular variant of thyroid papillary carcinoma | |
| Rana et al. | Expression of p53 and p16 in carcinoma breast tissue: Depicts prognostic significance or coincidence | |
| Egevad et al. | Interobserver reproducibility of perineural invasion of prostatic adenocarcinoma in needle biopsies | |
| Manenq et al. | Combined use of multiparametric flow cytometry and cytomorphology to enhance detection of neuroblastoma metastatic cells in bone marrow | |
| Ferronika et al. | p63 cytoplasmic aberrance is associated with high prostate cancer stem cell expression | |
| Vargas et al. | DNA damage‐inducible transcript 3 immunohistochemistry is highly sensitive for the diagnosis of myxoid liposarcoma but care is required in interpreting the significance of focal expression | |
| Eide et al. | Disseminated tumour cells in bone marrow of patients with uveal melanoma | |
| Riber-Hansen et al. | Extensive pathological analysis of selected melanoma sentinel lymph nodes: high metastasis detection rates at reduced workload | |
| Liao et al. | Establishment and characterization of patient-derived xenograft model of non-small-cell lung cancer derived from malignant pleural effusions | |
| CN110139656A (en) | The Keratin 17 of biomarker as bladder cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151111 |
|
| RJ01 | Rejection of invention patent application after publication |