CN103487583A - Immunohistochemical diagnosis kit for colorectal cancer malignancy and metastasis of colorectal cancer - Google Patents
Immunohistochemical diagnosis kit for colorectal cancer malignancy and metastasis of colorectal cancer Download PDFInfo
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Abstract
The invention discloses an immunohistochemical diagnosis kit for colorectal cancer malignancy and metastasis of colorectal cancer. The kit comprises an endogenous peroxidase inhibitor, a non-specific binding site inhibitor, a primary antibody, a secondary antibody, an enzyme and a color developing agent. The immunohistochemical diagnosis kit is characterized in that the primary antibody is a human S100A8 monoclonal antibody or a murine S100A8 monoclonal antibody, and the secondary antibody is biotin-labeled goat anti-mouse IgG or rabbit anti-mouse IgG. According to the kit, expressions of S100A8 antigens on the surface of colorectal pathological tissues are detected, so that the colorectal cancer malignancy and metastasis conditions can be rapidly and accurately judged, and a help is provided for clinical diagnosis of the colorectal cancer.
Description
Technical field
The present invention relates to the immunologic diagnosis of tumor kit, be specifically related to for colorectal cancer SABC kit.
Background technology
The calbindin family that S100 albumen is comprised of small molecular weight protein (10-14 kDa) can be dissolved in 100% saturation degree ammonium sulfate and gain the name because of it under the neutral pH condition.The research discovery, this albuminoid is only expressed in spinal animals, has found at present 23 kinds of S100 protein family members.S100A8 albumen is one of important member of S100 protein family, has conservative EF hand-type structure and the 4-helical annular of S100 protein family characteristic, by with Ca
2+, Zn
2+and Cu
2+deng the variation of different metal ions binding recurring structure, expose with the binding site of target protein and then by the biological effects such as participation inflammatory reaction, immune response and tumor development that interact with corresponding effect target protein.Although research shows both at home and abroad, S100A8 is remarkable high expressed in cancer of the stomach, colon cancer, prostate cancer, breast cancer, but for the relation of S100A8 albumen and specific tumors genesis, with and the application of S100A8 in the specific tumors diagnoses and treatment, still lack more deep experiment and research.
Colorectal cancer (colorectal cancer, CRC) be the common malignant tumour of alimentary canal, according to international cancer mechanism (IARC) Research statistics in 2008, in world wide, approximately have a million people that colorectal cancer occurs every year, whole world colorectal cancer mortality ratio accounts for tumour general mortality rate 8%, the tumour fatal rate is number four, and China's colorectal cancer case fatality rate is up to 45.5%, and generation and tumour that the grade malignancy of colorectal carcinoma and invasion and attack are shifted are lethal closely related.Therefore, find the detection method of a kind of quick judgement colorectal cancer grade malignancy and DISTANT METASTASES IN most important.
Summary of the invention
The object of the present invention is to provide the immunologic combined detection reagent kit of a kind of grade malignancy of Colorectal Cancer Diagnosis quickly and easily and DISTANT METASTASES IN, with SABC kit of the present invention, detect the expression of knot rectum pathological tissue surface S100A8 antigen, with this, can judge rapidly and accurately colorectal cancer grade malignancy and transfer case.
To achieve the above object of the invention, the inventor provides the immunologic combined detection reagent kit of a kind of Colorectal Cancer Diagnosis grade malignancy and DISTANT METASTASES IN, and this kit comprises: endogenous peroxydase blocking agent, nonspecific binding site blocking agent, primary antibodie, two anti-, enzyme, developer.The anti-S100A8 monoclonal antibody of the anti-S100A8 monoclonal antibody in described primary antibodie behaviour source or mouse source, described two resist for biotin labeled sheep anti-mouse igg or rabbit anti-mouse igg.
In the present invention, conventional various endogenous peroxydase blocking agent, nonspecific binding site blocking agent, enzyme, developers for the SABC test all can be used for test box of the present invention, but, as optimal selection, the endogenous peroxydase blocking agent described in the present invention is 3 wt%H preferably
2o
2, described nonspecific binding site blocking agent is preferably non-immune sheep serum, the preferred streptavidin peroxidase of described enzyme, the preferred DAB developer of described developer, comprise: DAB damping fluid (20 *), DAB substrate (20 *), DAB chromogen (20 *).
Further, optimization as kit of the present invention, also in kit, be provided for the comparison colour atla of reference, to facilitate, the coloration result of sample to be tested is compared, perhaps arrange staining cell percentage in the sample to be tested section is carried out to the counter automatically calculated, so that the sample dyeing result is done to express-analysis.
The inventor finds by research: the expression of S100A8 in colorectal cancer is apparently higher than normal colorectal carcinoma, and, there is marked difference in the expression of S100A8 albumen in different differentiation degree Expression in Colorectal Cancer intensity, in high, medium and low differentiation Colorectal Carcinoma, the strong positive rate of S100A8 protein expression is and obviously increases progressively trend, and differentiation degree is lower, and the strong positive expression is higher, differentiation degree is higher, and expression is lower; Simultaneously, in shifting Colorectal Carcinoma, S100A8 protein positive and strong positive expression are apparently higher than non-metastatic carcinoma tissue.Therefore, detect the expression of experimenter's colorectal carcinoma surface S100A8, can be used as the grade malignancy of colorectal cancer, the molecular marker of DISTANT METASTASES IN diagnosis.Therefore, the inventor provides above-mentioned SABC kit, utilizes this kit, ties the expression of rectum pathological tissue surface S100A8 antigen by detection, can judge rapidly and accurately colorectal cancer grade malignancy and transfer case, for the clinical diagnosis of colorectal cancer is offered help.
The accompanying drawing explanation
Fig. 1: analyze the corresponding S100A8 of NCBI GEO DataSets series GSE9348 middle probe 202917_s_at at the rna expression of the normal colorectal carcinomas of 12 example and 70 routine Colorectal Carcinomas figure as a result.
Fig. 2: SABC detects the expression of results figure of S100A8 albumen in normal colorectal carcinoma and high, medium and low differentiation Colorectal Carcinoma, and A-F figure is under microscope and amplifies the organization chart of 200 times, wherein:
A figure: normal colorectal carcinoma negative control (not adding the S100A8 primary antibodie);
B figure: Colorectal Carcinoma negative control (not adding the S100A8 primary antibodie);
C figure: normal colorectal carcinoma;
D figure: differentiated Colorectal Carcinoma;
E figure: middle differentiation Colorectal Carcinoma;
F figure: low differentiation Colorectal Carcinoma.
Fig. 3: SABC detects non-metastatic carcinoma and shifts the expression of results figure of S100A8 albumen in Colorectal Carcinoma, and following A, B figure are under microscope and amplify the organization chart of 200 times, wherein:
A figure: non-transfer Colorectal Carcinoma;
B figure: shift Colorectal Carcinoma.
Fig. 4: the IHC result that S100A8 albumen is expressed in normal colorectal carcinoma and cancerous tissue relatively.
Fig. 5: S100A8 albumen in the IHC of the Expression in Colorectal Cancer of different differentiation degrees result relatively.
Fig. 6: the IHC result that S100A8 albumen is expressed in the knot non-metastatic carcinoma of rectum and metastatic carcinoma relatively.
Embodiment
The inventor utilizes NCBI high flux gene expression data base GEO DataSets (gene expression omnibus), 70 routine Colorectal Carcinomas in serial GSE9348 and the normal colorectal carcinoma of 12 examples are carried out to S100A8 rna level chip data analysis, and result as shown in Figure 1.By this research, find: the expression of S100A8 in colorectal cancer be apparently higher than normal colorectal carcinoma, p<0.05, and difference has statistical significance.The inventor shows by the SABC further experiment: the expression of S100A8 in colorectal cancer is apparently higher than normal knot rectum group, and there is marked difference in the expression of S100A8 albumen in different differentiation degree Expression in Colorectal Cancer intensity, in high, medium and low differentiation Colorectal Carcinoma, the strong positive rate of S100A8 protein expression is and obviously increases progressively trend, be that differentiation degree is lower, the strong positive expression is higher, and differentiation degree is higher, and expression is lower; Simultaneously, in shifting Colorectal Carcinoma, S100A8 protein positive and strong positive expression are apparently higher than non-metastatic carcinoma tissue.Therefore, detect the expression of experimenter's colorectal carcinoma surface S100A8, can be used as the grade malignancy of colorectal cancer, the molecular marker of DISTANT METASTASES IN diagnosis.
Based on above-mentioned result of study, the present invention prepares the immunologic combined detection reagent kit of a kind of Colorectal Cancer Diagnosis grade malignancy and DISTANT METASTASES IN, and this kit comprises: endogenous peroxydase blocking agent, nonspecific binding site blocking agent, primary antibodie, two anti-, enzyme, developer.The anti-S100A8 monoclonal antibody in described primary antibodie behaviour source or mouse source S100A8 monoclonal antibody, described two resist for biotin labeled sheep anti-mouse igg or rabbit anti-mouse igg.
Conventional various endogenous peroxydase blocking agent, nonspecific binding site blocking agent, enzyme, developers for the SABC test all can be used for test box of the present invention, but, as optimal selection, in the present embodiment, the endogenous peroxydase blocking agent is 3 wt%H
2o
2, described nonspecific binding site blocking agent is non-immune sheep serum, described enzyme is streptavidin peroxidase, described developer is the DAB developer, comprise: DAB damping fluid (20 *), DAB substrate (20 *), DAB chromogen (20 *).
Be provided for the comparison colour atla of reference in kit, be respectively faint yellow, yellow and sepia.And a scan counter is set, this counter comprises that an energy carries out scanning element to the displaing micro picture of section, and comprises one scanned picture is carried out to Cytometric analytic unit.Use this counter, can treat staining cell percentage in institute's this section of test sample and automatically calculate, so that the sample dyeing result is done to express-analysis.
the kit of embodiment 2 use embodiment 1 preparations detects the Colorectal Carcinoma sample
(1) roasting sheet dewaxing: get colorectal cancer patients tissue, the histotomy wait dyeing is put into to 60 ℃ of baking boxs bakings and put into rapidly dimethylbenzene (I, II) immersion in 60-90 minute after (depending on chipping qualities) and dewaxed for 10 minutes * 2 times;
(2)------PBS washing that 50% ethanol soaks 5 minutes--distilled water soaks 3 minutes--3 minutes * 3 times that 75% ethanol soaks 5 minutes that 90% ethanol soaks 5 minutes that aquation: section is placed in order with Gradient alcohol: 100% ethanol soaks 5 minutes;
(3) antigen retrieval: the citric acid antigen retrieval liquid of 0.01M pH6.0 boils in micro-wave oven, section is put into wherein, emgloves seals vessel port, 15-20 minute is heated in continuation in boiling water, make temperature remain on 100 ℃, complete container naturally cools to room temperature, will not cut into slices and take out from repair liquid, after use again on 1 * PBS damping fluid shaking table rinsing 3 minutes * 3 times;
(4) blocking-up endogenous peroxydase: section is placed in and hatches box, with wax crayon, tissue is enclosed, in order to avoid the diffusion of reagents after dripping, thieving paper slightly sucks the water in section, does not encounter tissue, uses 3 wt%H
2o
2solution 100 μ l cover after tissue incubated at room 30 minutes, to remove endogenous peroxydase, then use on 1 * PBS damping fluid shaking table rinsing 3 minutes * 3 times;
(5) nonspecific binding site serum sealing: thieving paper slightly sucks the water in section, drips non-immune sheep serum, and 100 μ l, seal 30 minutes, gets rid of gently supernatant, not rinsing;
(6) primary antibodie is hatched: in hatching box, drip the anti-S100A8 monoclonal antibody solution in mouse source (purchased from ABNOVA company) after 1 * PBS dilutes; 1:200 dilution, every section is 100 μ l approximately, hatch 1-2 hour for 37 ℃, or 4 ℃ spent the night, then 1 * PBS damping fluid shaking table upper 3 minute * 3 rinsings.Negative control adopts PBS to replace primary antibodie.；
(7) two anti-hatching: thieving paper slightly sucks the water in section, and biotin labeled sheep anti-mouse igg 100 μ l(step new purchased from Foochow), room temperature 15 minutes, 1 * PBS damping fluid shaking table upper 3 minute * carry out rinsing 3 times;
(8) thieving paper slightly sucks the water in section, and every section drips streptavidin peroxidase 100 μ l, incubated at room 15 minutes, then use 1 * PBS damping fluid shaking table upper 3 minute * carry out rinsing 3 times;
(9) colour developing and termination: thieving paper slightly sucks the water in section, dry plate not, dropping DAB nitrite ion DAB damping fluid (20 *), DAB substrate (20 *), each 50 μ l of DAB chromogen (20 *) drop in 850 μ l distilled waters and are mixed with the fresh demonstration liquid of 1mL, effective storage life is developed the color in 30 minutes, controlling developing time under microscope is 60 seconds, dye to brown color or tawny cessation reaction in complete tap water;
(10) redye: thieving paper slightly sucks the water in section, drips haematoxylin and redyes 15 seconds, rinses cessation reaction with tap water rapidly, and tap water returns blue 5-10 minute;
(11) dewater transparent: enter successively each 2 minutes fast dewaterings of 50 wt% ethanol, 75 wt% ethanol, 90 wt% ethanol, 100 wt% ethanol after drying.Reentering in dimethylbenzene 2 minutes carries out transparent;
(12) mounting: after slice dries naturally, drip neutral gum and cover glass mounting.
With the kit of above-described embodiment 1, according to method and the step of embodiment 2, detected the expression of S100A8 albumen in the normal and 104 routine colorectal cancer gross specimens of 54 examples.All tissue specimens carry out proved by pathology in operation by pathology department of Xiangye No. 2 Hospital of Central South University; In 104 routine colorectal carcinoma samples, 58 examples are non-metastatic carcinoma, and 46 examples are the DISTANT METASTASES IN cancer; Wherein 96 examples can be used for the grade malignancy analysis: 14 routine well-differentiated carcinomas, break up cancer in 48 examples, 34 routine poor differentiated carcinomas.
In intracellular location, carry out comprehensive grading with protein expression positive signal intensity and two kinds of standards of positive expression cell number according to albumen respectively.Judgement positive expression result standard: (1) judges according to positive staining intensity: cell dye-free note 0 minute; Cell is dyed faint yellow, remembers 1 minute; Yellow, remember 2 minutes; Sepia, remember 3 minutes.(2) expressing number scale according to positive cell divides: positive cell number<5%, remember 0 minute; 5% ~ 25%, remember 1 minute; 26% ~ 50%, remember 2 minutes; 51% ~ 75%, remember 3 minutes, 75%, remember 4 minutes.Final score result is the addition of above-mentioned two kinds of criterion scores.Outcome evaluation: 0 is divided into negative express (-); 1,2 are divided into weak positive expression (+); 3 ~ 5 are divided into the medium positive (+); 6,7 are divided into strong positive expression (+).The result of analyzing S100A8 SABC in tissue shows, S100A8 albumen is at 97.12%(101/104) colorectal cancer and 37.93%(22/58) normal colorectal carcinoma in express positive, see Fig. 4 and Fig. 2, in Colorectal Carcinoma the S100A8 protein expression level apparently higher than normal colorectal carcinoma (
x 2 =
? 68.15, p=0.00).Kruskal-Wallis H inspection statistics analysis discovery, the expression of S100A8 albumen has difference in different differentiation degree Expression in Colorectal Cancer intensity,
x 2 =7.73, P=0.021, difference has statistical significance.High, medium and low differentiation cancer strong positive rate is respectively 14.28%, 43.75% and 61.76%, is and obviously increases progressively trend, and differentiation degree is lower, and the strong positive expression is higher, sees Fig. 5 and Fig. 2; And in the metastatic carcinoma tissue, S100A8 protein positive and strong positive expression apparently higher than non-metastatic carcinoma tissue (
x 2 =4.21, P=0.04), see Fig. 6 and Fig. 3, difference has statistical significance.
Further clinical practice shows, thereby kit of the present invention can degree of tissue differentiation and the transfer case of colorectal cancer contribute to Colorectal Cancer Diagnosis quickly and easily for accurately judging by detecting Colorectal Carcinoma S100A8 protein expression level.
Should be understood that, after having read above-mentioned disclosure of the present invention, those skilled in the art can make various changes or modification to the present invention, these equivalent form of values fall within claims limited range of the application equally.
Claims (6)
1. the immunodiagnosis reagent kit of colorectal cancer grade malignancy and transfer thereof, this kit comprises: endogenous peroxydase blocking agent, nonspecific binding site blocking agent, primary antibodie, two anti-, enzyme, developer, it is characterized in that the anti-S100A8 monoclonal antibody of the anti-S100A8 monoclonal antibody in described primary antibodie behaviour source or mouse source, described two resist for biotin labeled sheep anti-mouse igg or rabbit anti-mouse igg.
2. kit according to claim 1, it is characterized in that: described endogenous peroxydase blocking agent is 3wt%H
2o
2, described nonspecific binding site blocking agent is nonimmune animal blood serum, and described enzyme is streptavidin peroxidase, and described developer is the DAB developer.
3. kit according to claim 1 and 2, it is characterized in that: described nonspecific binding site blocking agent is non-immune sheep serum.
4. one of the described kit according to claim 1-3 is characterized in that: the comparison colour atla of the reference that is provided in described kit dyeing, described comparison colour atla has three, is respectively faint yellow, yellow and sepia.
5. one of the described kit according to claim 1-3, is characterized in that: arrange in kit staining cell percentage in the sample to be tested section is carried out to the counter automatically calculated.
6. kit according to claim 4, is characterized in that: arrange in kit staining cell percentage in the sample to be tested section is carried out to the counter automatically calculated.
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Cited By (3)
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| CN104880565A (en) * | 2015-05-04 | 2015-09-02 | 贵州省人民医院 | Detection reagent for prognosis of ZFP36 prostatic cancer and kit of detection reagent |
| CN105891484A (en) * | 2016-05-06 | 2016-08-24 | 中南大学湘雅医院 | Group of colon cancer metastasis related tumor stroma markers and application |
| CN112710526A (en) * | 2019-10-24 | 2021-04-27 | 广州江元医疗科技有限公司 | Cell fixing and preserving fluid and preparation method and application thereof |
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Application publication date: 20140101 |