CN110095605B - Lung cancer screening kit - Google Patents
Lung cancer screening kit Download PDFInfo
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- CN110095605B CN110095605B CN201910347649.3A CN201910347649A CN110095605B CN 110095605 B CN110095605 B CN 110095605B CN 201910347649 A CN201910347649 A CN 201910347649A CN 110095605 B CN110095605 B CN 110095605B
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- 206010058467 Lung neoplasm malignant Diseases 0.000 title claims abstract description 45
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- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 26
- 238000001514 detection method Methods 0.000 claims description 12
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- 102000036639 antigens Human genes 0.000 description 8
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- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
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- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
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- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The invention discloses a lung cancer screening kit, which comprises an optional reagent for detecting the expression level of phosphorylated BLK. The invention also discloses application of the reagent for detecting the expression level of the phosphorylated BLK in preparing a reagent for screening lung cancer. The kit can effectively judge the risk of lung cancer of the people to be detected by detecting the expression level of the phosphorylated BLK, can be used for auxiliary diagnosis of clinical lung cancer, provides effective basis for patients to take relevant treatment measures or decisions, and has good clinical application prospect.
Description
Technical Field
The invention relates to a lung cancer screening kit.
Background
Lung cancer is one of the most rapidly growing malignancies that threaten human health and life. In many countries, the incidence and mortality of lung cancer have been reported to be significantly higher in recent 50 years, with lung cancer incidence and mortality in men accounting for the first of all malignancies, in women accounting for the second, and mortality accounting for the second. Non-small cell lung cancer is a group of lung cancers that account for approximately 80% of all lung cancers, with approximately 75% of patients found in the middle and advanced stages with very low 5-year survival rates. Therefore, it is very important to diagnose lung cancer rapidly and early.
The screening of lung cancer refers to the routine examination of people without lung cancer related symptoms and lung cancer is found before symptoms appear. If the lung cancer molecular marker in blood can be found, the molecular marker has important significance for prompting a clinician to take relevant treatment measures or decisions for a patient at an early stage.
At present, the diagnosis of lung cancer mostly depends on the biopsy of lung tissues, but the detection method has the disadvantages of complex operation, great lung injury to patients, and great risk of lung cancer diffusion. The detection method which is more simple, convenient, small in wound and small in side effect needs to be solved urgently.
Phosphorylated BLK, a phosphorylated B lymphocyte tyrosine kinase. There is no literature report on the detection of lung cancer by detecting phosphorylated BLK.
Disclosure of Invention
In order to solve the problems, the invention provides a lung cancer detection kit which is small in wound, small in side effect and convenient to use.
The lung cancer screening kit of the present invention includes an optional reagent for detecting the expression level of phosphorylated BLK.
Wherein the reagent is a reagent for detecting the expression level of phosphorylated BLK in serum and/or lung tissue.
Wherein the reagent for detecting the expression level of phosphorylated BLK is a reagent for ELISA detection.
Wherein the reagent for detecting the expression level of the phosphorylated BLK is a reagent for a Western-blot detection method.
The invention also provides application of the reagent for detecting the expression level of the phosphorylated BLK in preparing a reagent for screening lung cancer.
Wherein the reagent for detecting the expression level of phosphorylated BLK is a reagent for ELISA detection.
Wherein the reagent for detecting the expression level of the phosphorylated BLK is a reagent for a Western-blot detection method.
Wherein the reagent is a reagent for detecting the expression level of phosphorylated BLK in serum and/or lung tissue.
The kit can judge the risk of lung cancer of the people to be detected by detecting the expression level of the phosphorylated BLK, can be used for auxiliary diagnosis of clinical lung cancer, can screen the risk of lung cancer only by collecting blood of a sample to be detected, and has the advantages of small wound, small side effect, convenient use and good clinical application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Detailed Description
Example 1 correlation of expression levels of phosphorylated BLK with Lung cancer
First, experiment method
1. Clinical data
30 paraffin sections of lung cancer patients and 30 far-end controls (the paraffin section of far-end normal lung tissue of the lung cancer patients is 5cm away from cancer tissues and is confirmed to be normal tissues pathologically) are selected, and the basic information is shown in table 1.
TABLE 1 clinical basic information
*Note: others include large cell carcinoma, adenosquamous carcinoma, small cell carcinoma.
Paraffin sections of lung cancer patients and paraffin sections of distal tissue controls were subjected to Immunohistochemistry (IHC) using phosphorylated BLK as an index.
Phosphorylated BLK antibodies: available from Affinity, usa under cat # AF 8133;
EnVisionTMHRP secondary antibody, antibody diluent, DAB color developing agent, antigen repairing liquid, Wash Buffer and immunohistochemical pen: all available from Dako, denmark.
The expression level of phosphorylated BLK was measured as follows:
(1) blocking peroxidase: dewaxing paraffin section to hydrate (dewaxing process: xylene I10min → xylene II10min → absolute ethanol 5min → 95% ethanol 5min → 85% ethanol 5min → 75% ethanol 5min), adding 3% H2O2Sealing endogenous peroxidase in the dark, and incubating at room temperature for 15 min;
(2) rinsing with distilled water: placing the slices in distilled water, and washing on a shaking table for 5 min/time for three times;
(3) antigen retrieval: putting the slices into EDTA antigen retrieval solution with pH of 9.0, and carrying out water bath in a water bath kettle at 98 ℃ for 40min to carry out antigen retrieval;
(4) rinsing with distilled water: taking out the slices, naturally cooling to room temperature, washing with distilled water for three times, and rinsing with WashBuffer once;
(5) incubating the primary antibody: enclosing the tissue by using an immunohistochemical pen, dripping a phosphorylated BLK antibody working solution (the phosphorylated BLK is mixed with an antibody diluent at a ratio of 1: 150) into the enclosure, and incubating overnight at 4 ℃;
(6) rinsing with distilled water: washing with distilled water for three times, and rinsing with Wash Buffer for one time;
(7) and (4) dropwise adding a secondary antibody: dripping Dako HRP labeled secondary antibody working solution, and incubating for 60min at room temperature;
(8) rinsing with distilled water: washing with distilled water for 5min for 3 times;
(9) DAB color development: dropwise adding a chromogenic substrate DAB, observing the chromogenic condition under a microscope, and terminating the reaction in distilled water;
(10) hematoxylin counterstaining: placing the slices in hematoxylin staining solution for staining for 2min, and washing with running water for 10min after differentiation of 1% hydrochloric acid alcohol;
(11) and (3) dehydrating and transparency: dehydrating the slices by 75%, 85%, 95% and 100% gradient alcohol, making the slices transparent by xylene, and drying the slices in a fume hood;
(12) sealing: and (4) using a neutral quick-drying adhesive sealing sheet, observing under an optical microscope, and acquiring a picture by a DP Controller image acquisition system. After image acquisition, immunohistochemical scores were given based on tumor cell staining intensity and positive area in lung cancer patients and distant tissues.
3. Immunohistochemical scoring criteria
The tumor cell staining intensity, positive area score were first specified as follows:
@note: two experienced pathologists independently scored the tumor cell staining intensity results
The product of the tumor cell staining intensity score and the positive area score (0-9 points) was then calculated, specifying:
phosphorylated BLK expression negative: 0 minute;
phosphorylated BLK expresses low positivity: 1-3 min;
phosphorylated BLK expression was highly positive > 3 points.
4. Analysis of results
Statistical analysis was performed using SPSS17.0 lung cancer tissue group and remote tissue control group.
Second, experimental results
The results of the expression level measurements of phosphorylated BLK in lung cancer tissue versus distant tissue controls are shown in table 2.
TABLE 2 expression levels of phosphorylated BLK
As can be seen from Table 2, the positive expression rate of phosphorylated BLK in the distal normal tissue is 6.7%, and the high positive expression rate is 0%; the positive expression rate of phosphorylated BLK in lung cancer tissues is 93.3 percent, and the high positive expression rate is 60 percent; phosphorylated BLK was significantly elevated in lung cancer tissues, and the difference in the expression level of phosphorylated BLK was statistically significant (P < 0.0001) compared to distant normal tissues.
From the results, the expression level of phosphorylated BLK in the lung cancer tissue is obviously increased (P is less than 0.0001) compared with the paracancer normal lung tissue, which indicates that the expression level of the phosphorylated BLK is in positive correlation with the lung cancer, and the high expression of the phosphorylated BLK can obviously improve the possibility of suffering from the lung cancer. Since the level of phosphorylated BLK in the paracancerous normal lung tissue can reflect the level of phosphorylated BLK in the normal human lung tissue, the susceptible population of lung cancer can be screened by detecting the expression level of phosphorylated BLK of a patient to be detected.
Example 2 Lung cancer screening kit and methods of use
Kit composition
Detection kit (50 parts):
| components | Volume of |
| Phosphorylated BLK antibodies | 100μl |
| EnVisionTMHRP secondary antibody | 10ml |
| Antibody diluent | 10ml |
| DAB color-developing agent | 10ml |
| Antigen retrieval liquid | 500ml |
| Wash Buffer* | 500ml |
| Immunohistochemical pen | 1 count |
Note: the kit is counted by 100 mul of each experimental reagent dripped into each section, and the specific dosage can be properly increased and decreased according to the size of the tissue.
Second, using method of kit
Preparing a paraffin section from lung tissues of a sample to be detected, and detecting the expression level of phosphorylated BLK as a detection specimen according to the following method:
(1) blocking peroxidase: dewaxing paraffin section until hydration (the dewaxing process is xylene I10min → xylene II10min → absolute ethanol 5min → 95% ethanol 5min → 85% ethanol 5min → 75% ethanol 5min), sealing endogenous peroxidase with 3% H2O2 solution in dark, and incubating at room temperature for 15 min;
(2) rinsing with distilled water: placing the slices in distilled water, and washing on a shaking table for 5 min/time for three times;
(3) antigen retrieval: putting the slices into EDTA antigen retrieval solution with pH of 9.0, and carrying out water bath in a water bath kettle at 98 ℃ for 40min to carry out antigen retrieval;
(4) rinsing with distilled water: taking out the slices, naturally cooling to room temperature, washing with distilled water for three times, and rinsing with WashBuffer once;
(5) incubating the primary antibody: enclosing the tissue with an immunohistochemical pen, dripping a phosphorylated BLK antibody working solution (the phosphorylated BLK is mixed with an antibody diluent at a ratio of 1: 150) in the enclosure, and incubating overnight at 4 ℃;
(6) rinsing with distilled water: washing with distilled water for three times, and rinsing with Wash Buffer for one time;
(7) and (4) dropwise adding a secondary antibody: dripping Dako HRP labeled secondary antibody working solution, and incubating for 60min at room temperature;
(8) rinsing with distilled water: washing with distilled water for 5min for 3 times;
(9) DAB color development: dropwise adding a chromogenic substrate DAB, observing the chromogenic condition under a microscope, and terminating the reaction in distilled water;
(10) hematoxylin counterstaining: placing the slices in hematoxylin staining solution for staining for 2min, and washing with running water for 10min after differentiation of 1% hydrochloric acid alcohol;
(11) and (3) dehydrating and transparency: dehydrating the slices by 75%, 85%, 95% and 100% gradient alcohol, making the slices transparent by xylene, and drying the slices in a fume hood;
(12) sealing: and (4) using a neutral quick-drying adhesive sealing sheet, observing under an optical microscope, and acquiring a picture by a DP Controller image acquisition system. After the image is taken, immunohistochemical scores are given according to the staining intensity and positive area of tumor cells of lung cancer patients and distant tissues, and the scoring method is shown in example 1.
For abnormal lung, abnormal part and normal part tissues can be respectively taken, and the expression level of phosphorylated BLK is compared, so that the possibility of lung cancer is evaluated, and the method is used as an auxiliary diagnosis means for clinical lung cancer.
In conclusion, the kit can screen the risk of the people to be detected to suffer from the lung cancer by detecting the expression level of the phosphorylated BLK, can be used for the auxiliary diagnosis of clinical lung cancer, provides effective basis for patients to take related treatment measures or decisions, and has good clinical application prospect.
Claims (3)
1. The use of a reagent for detecting the expression level of phosphorylated BLK in the preparation of a reagent for screening lung cancer;
the reagent for detecting the expression level of the phosphorylated BLK comprises a phosphorylated BLK antibody, wherein the antibody is a phosphorylated BLK antibody with the commodity number of AF8133 of the Affinity company in the United states;
when the fact that the expression level of phosphorylated BLK of a lung tissue sample of a patient to be detected is obviously higher than that of a normal lung tissue is detected, the possibility that the patient to be detected suffers from lung cancer is high.
2. Use according to claim 1, characterized in that: the reagent for detecting the expression level of phosphorylated BLK is a reagent for ELISA detection.
3. Use according to claim 1, characterized in that: the reagent for detecting the expression level of the phosphorylated BLK is a reagent for a Western-blot detection method.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012125712A2 (en) * | 2011-03-14 | 2012-09-20 | Respira Health, Llc | Lung tumor classifier for current and former smokers |
| CN106918698A (en) * | 2015-12-25 | 2017-07-04 | 广州瑞博奥生物科技有限公司 | A kind of phospho-AB chip agent box for detecting human receptor tyrosine kinase enzyme |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012125712A2 (en) * | 2011-03-14 | 2012-09-20 | Respira Health, Llc | Lung tumor classifier for current and former smokers |
| CN106918698A (en) * | 2015-12-25 | 2017-07-04 | 广州瑞博奥生物科技有限公司 | A kind of phospho-AB chip agent box for detecting human receptor tyrosine kinase enzyme |
Non-Patent Citations (1)
| Title |
|---|
| Distinct expressions of immunoglobulin signal related molecules between human Kawasaki disease and BALB/c mice treated with Lactobacillus casei cell wall extract;Hong-Ren Yu et al;《Int J Clin Exp Med》;20161030;第9卷(第10期);19502-19511 * |
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