CN111812328A - Kit for early detection and diagnosis of liver cancer metastasis - Google Patents
Kit for early detection and diagnosis of liver cancer metastasis Download PDFInfo
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Abstract
The invention belongs to the field of biological medicine, and particularly relates to a liver cancer metastasis early detection and diagnosis kit, which comprises a mouse anti-human S100A11 monoclonal antibody, confining liquid, working liquid, antibody diluent, goat anti-human secondary antibody, DAB developing liquid, 0.01mol/L sodium citrate buffer solution, hematoxylin dyeing liquid, neutral resin, 70% ethanol, 75% ethanol, 80% ethanol, 95% ethanol, absolute ethanol, xylene and PBS; the kit can judge the difference of the expression levels of the S100A11 protein of the abnormal liver by detecting the expression levels of the S100A11 protein of the abnormal liver part and the normal liver part of the abnormal liver part, thereby judging the risk of liver cancer of a person to be detected.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the field of biological medicines, and particularly relates to a kit for early detection and diagnosis of liver cancer metastasis.
[ background of the invention ]
Liver cancer can be roughly classified into 2 types, one is primary liver cancer, and the other is metastatic liver cancer. Primitive liver cancer is a cancer that grows on the surface of the liver itself; however, metastatic liver cancer gradually migrates to the liver due to cancer cells of other tissues and organs, and thus gradually develops into liver cancer. Generally, the primary liver cancer will progress to the late stage, which is much longer than the metastatic liver cancer, and generally, the primary liver cancer will gradually progress to the liver cancer after about 15 years.
In patients with advanced liver cancer, when advanced cancer is found, it means that cancer cells in the body have spread in large numbers, and the longer the period, the higher the degree of deterioration, and some people can live even in a few months. Therefore, the time of diagnosis plays a decisive factor. However, at present, no kit for rapidly, simply and conveniently detecting and diagnosing liver cancer metastasis at an early stage is available, so that the patient cannot be treated in time after the diagnosis time of the liver cancer is delayed.
The S100a11 protein is a member of the calcium binding protein S100 family of twenty or more members, and as with most S100 proteins, Ca2+ binding causes a dramatic change in its conformation to interact with its various target proteins. In this family, Ca2+ flux is a signal that regulates a variety of cellular processes, including apoptosis, contraction, differentiation, gene expression, and many other physiological processes. Target proteins of S100A1 include Ca2+ signal transduction proteins, neurotransmitter release-related proteins (synapsin I, II), cytoskeletal and filament junction proteins, transcription factors and their regulators, enzymes, and other Ca2+ activator proteins, etc. S100a11 functions both as a signaling molecule and as a secreted protein in a cell.
[ summary of the invention ]
In view of the above problems, the present invention provides a kit for early detection and diagnosis of liver cancer metastasis.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
an early detection and diagnosis kit for liver cancer metastasis comprises a mouse anti-human S100A11 monoclonal antibody, a confining liquid, a working solution, an antibody diluent, a goat anti-human second antibody, a DAB developing solution, a sodium citrate buffer solution with the concentration of 0.01mol/L, a hematoxylin staining solution, neutral resin, 70% ethanol, 75% ethanol, 80% ethanol, 95% ethanol, absolute ethanol, xylene and PBS;
the confining liquid is a 5% bovine serum albumin solution prepared by using phosphate buffer PBS as a solvent; the working solution is prepared by diluting 30% H with PBS solution2O2Solution dilution to 3% H2O2A solution; the antibody diluent comprises 0.1% (V/V) Tween-20 phosphate and 0.1MPBS solution with pH7.4.
An application method of a kit for early detection and diagnosis of liver cancer metastasis comprises the following steps:
1) taking liver tissues of a person to be detected to carry out paraffin embedding slicing, placing the liver tissues in a 60 ℃ oven for 15min, and dewaxing the paraffin slices to water to obtain dewaxed slices;
2) placing the dewaxed slices in xylene for 10min, replacing xylene and soaking for 10min, then soaking in absolute ethanol for 5min, soaking in 95% ethanol for 5min, soaking in 80% ethanol for 5min, soaking in 75% ethanol for 5min, soaking in 70% ethanol for 5min, soaking in PBS for 5min, then repeatedly soaking in PBS for 3 times, and obtaining slices after primary treatment;
3) placing the slice after the preliminary treatment in a working solution for incubation for 20min at room temperature to eliminate the activity of endogenous peroxidase to obtain the slice after enzyme deactivation treatment;
4) washing the slices after enzyme deactivation with distilled water, then soaking for 5min with PBS, and repeatedly soaking for 3 times with PBS replaced to obtain washed slices;
5) placing the cleaned slices into a sodium citrate buffer solution with the concentration of 0.01mol/L, placing the slices into a microwave oven to be heated at the temperature of 90-99 ℃, then placing the slices at room temperature for 11-15min, and repeating the steps for 3 times to obtain the slices after antigen retrieval;
6) placing the sections subjected to antigen restoration in a sealing solution, incubating at room temperature for 20-30 min, removing the sealing solution, dropwise adding a mouse anti-human S100A11 monoclonal antibody into the sections, and incubating at 37 ℃ for 1-2 hours to obtain treated sections;
7) soaking the treated slices in PBS for 5min, and repeatedly soaking for 3 times by replacing PBS;
8) adding a goat anti-human secondary antibody marked by HRP (horse radish peroxidase) dropwise into the processed slices, wherein the goat anti-human secondary antibody is a product of KPL company, and incubating for 60min at 37 ℃ to obtain slices processed by the secondary antibody;
9) soaking the slices treated by the secondary antibody in PBS for 5min, and repeatedly soaking for 3 times by replacing PBS;
10) dripping a proper amount of working solution into the processed slices, and incubating for 60min at 37 ℃;
11) soaking the processed slices in PBS for 5min, and repeatedly soaking for 3 times by replacing PBS;
12) placing the processed slices in DAB developing solution for developing color at room temperature, observing the developing process of the slices under a microscope, controlling the reaction time to be 5-10 min, and washing the slices with distilled water to obtain the developed slices;
13) dropwise adding hematoxylin staining solution on the slices after the color development, slightly re-staining the slices, then differentiating the slices for 30s by using hydrochloric acid and alcohol, and washing the slices for 10min by using distilled water to obtain the re-stained slices;
14) soaking the slices after the redyeing in 75% ethanol for 5min and 80% ethanol for 5min, soaking in 95% ethanol for 5min and repeating for 3 times, soaking in 100% ethanol for 5min and repeating for 3 times, soaking in xylene for 20min and repeating for 3 times, and sealing the slices with neutral gum;
15) the microscope photographs the mounting film, and a DPController image acquisition system is used for acquiring images; after image acquisition, immunohistochemistry scores were given according to cell staining intensity and positive area.
Positive staining was indicated by the presence of a tan or yellow-brown particle in the nucleus and cytoplasm of the positively stained cells.
Preferably, the mouse anti-human S100A11 monoclonal antibody in the step 6) is prepared by diluting the mouse anti-human S100A11 monoclonal antibody with an antibody diluent, and the ratio of the mouse anti-human S100A11 monoclonal antibody to the antibody diluent is 1: 100.
Due to the adoption of the technical scheme, the invention has the following beneficial effects: the kit can judge the difference of the expression levels of the S100A11 protein of the abnormal liver by detecting the expression levels of the S100A11 protein of the abnormal liver part and the normal liver part of the abnormal liver part, and further judge the risk of liver cancer metastasis of a person to be detected: if the expression level of the S100A11 protein is high, the risk of liver cancer metastasis is high, and the metastasis of liver cancer cells of a patient needs to be monitored and prevented. If the expression level of the S100A11 protein is low, the risk of liver cancer metastasis is low.
[ description of the drawings ]
FIG. 1 is an optical microscopic image of human S100A11 protein cells.
[ detailed description ] embodiments
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that all the directional indicators (such as up, down, left, right, front, and rear … …) in the embodiment of the present invention are only used to explain the relative position relationship between the components, the movement situation, etc. in a specific posture (as shown in the drawing), and if the specific posture is changed, the directional indicator is changed accordingly.
In addition, the descriptions related to "first", "second", etc. in the present invention are for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, the meaning of "and/or" appearing throughout is to include three juxtapositions, exemplified by "A and/or B," including either the A or B arrangement, or both A and B satisfied arrangement. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
Examples
An early detection and diagnosis kit for liver cancer metastasis comprises a mouse anti-human S100A11 monoclonal antibody, a confining liquid, a working solution, an antibody diluent, a goat anti-human second antibody, a DAB developing solution, a sodium citrate buffer solution with the concentration of 0.01mol/L, a hematoxylin staining solution, neutral resin, 70% ethanol, 75% ethanol, 80% ethanol, 95% ethanol, absolute ethanol, xylene and PBS;
the confining liquid is prepared by using Phosphate Buffered Saline (PBS) as a solvent5% bovine serum albumin solution; the working solution is prepared by diluting 30% H with PBS solution2O2Solution dilution to 3% H2O2A solution; the antibody diluent comprises 0.1% (V/V) Tween-20 phosphate and 0.1MPBS solution with pH7.4.
Relationship between S100A11 protein expression level and liver cancer
Experimental methods
Selecting paraffin sections of 50 cases of liver cancer patients and far-end control 50 cases (referring to paraffin of far-end normal liver tissue of liver cancer patients)
Sliced 2-6cm from cancer tissue), the basic information is shown in table 1.
| Slice information | Liver cancer tissue group | Remote control group |
| Number of people | 20 | 20 |
| Age (age) | 58±10.5 | 59±11.5 |
| Proportion of male | 25(50％) | 25(50％) |
The expression level of the S100A11 protein is detected by using the liver cancer metastasis early detection and diagnosis kit according to the following method:
1) taking pathological tissues of a liver cancer patient and far-end normal liver tissues to carry out paraffin embedding slicing, placing the slices in a 60 ℃ oven for 15min, and dewaxing the paraffin slices to water to obtain the dewaxed slices;
2) placing the dewaxed slices in xylene for 10min, replacing xylene and soaking for 10min, then soaking in absolute ethanol for 5min, soaking in 95% ethanol for 5min, soaking in 80% ethanol for 5min, soaking in 75% ethanol for 5min, soaking in 70% ethanol for 5min, soaking in PBS for 5min, then repeatedly soaking in PBS for 3 times, and obtaining slices after primary treatment;
3) placing the slice after the primary treatment in 3% H2O2, and incubating for 20min at room temperature to eliminate the activity of endogenous peroxidase to obtain the slice after enzyme deactivation treatment;
4) washing the slices after enzyme deactivation with distilled water, then soaking for 5min with PBS, and repeatedly soaking for 3 times with PBS replaced to obtain washed slices;
5) placing the cleaned slices into a sodium citrate buffer solution with the concentration of 0.01mol/L, placing the slices into a microwave oven to be heated at the temperature of 90-99 ℃, then placing the slices at room temperature for 11-15min, and repeating the steps for 3 times to obtain the slices after antigen retrieval;
6) placing the sections subjected to antigen restoration in a 5% bovine serum albumin solution prepared by using PBS as a solvent, incubating for 20-30 min at room temperature, removing the 5% bovine serum albumin solution prepared by using PBS as a solvent, dropwise adding a mouse anti-human S100A11 monoclonal antibody into the sections, and incubating for 1-2 hours at 37 ℃ to obtain treated sections;
7) soaking the treated slices in PBS for 5min, and repeatedly soaking for 3 times by replacing PBS;
8) adding a goat anti-human secondary antibody marked by HRP (horse radish peroxidase) dropwise into the processed slices, wherein the goat anti-human secondary antibody is a product of KPL company, and incubating for 60min at 37 ℃ to obtain slices processed by the secondary antibody;
9) soaking the slices treated by the secondary antibody in PBS for 5min, and repeatedly soaking for 3 times by replacing PBS;
10) dripping a proper amount of working solution into the processed slices, and incubating for 60min at 37 ℃;
11) soaking the processed slices in PBS for 5min, and repeatedly soaking for 3 times by replacing PBS;
12) DAB color development: placing the processed slices in DAB for room temperature color development, observing the color development process of the slices under a microscope, controlling the reaction time to be 5-10 min, and washing the slices with distilled water to obtain the colored slices;
13) dropwise adding hematoxylin staining solution on the slices after the color development, slightly re-staining the slices, then differentiating the slices for 30s by using hydrochloric acid and alcohol, and washing the slices for 10min by using distilled water to obtain the re-stained slices;
14) soaking the slices after the redyeing in 75% ethanol for 5min and 80% ethanol for 5min, soaking in 95% ethanol for 5min and repeating for 3 times, soaking in 100% ethanol for 5min and repeating for 3 times, soaking in xylene for 20min and repeating for 3 times, and sealing the slices with neutral gum;
15) the microscope photographs the mounting film, and a DPController image acquisition system is used for acquiring images; after image collection, immunohistochemical scores were given based on the intensity of tumor cell staining and the positive area of the liver cancer patients and distal tissues.
Immunohistochemical scoring criteria
Tumor cell staining intensity-tumor cell positive area-0-9 points,
and (4) counting results: negative: 0 minute; low degree of positivity: 1-3 min; highly positive >3 points.
Wherein, the staining intensity and positive area of the tumor cells are scored as follows:
immunohistochemical staining results were scored independently by two experienced pathologists
4. Analysis of results
The liver cancer tissue group and the remote tissue control group were subjected to statistical analysis.
Second, experimental results
The results of the expression level detection of the S100a11 protein in the liver cancer tissue group and the remote tissue control are shown in table 2.
| Sample (I) | Negative of | Low positive | High positive |
| Liver cancer tissue group | Example 1 | 23 example(s) | 26 examples of |
| Distal tissue control group | 49 examples of | Example 1 | Example 0 |
TABLE 2 expression level of S100A11 protein
As can be seen from table 2, the positive expression rate of the S100a11 protein in the distal tissue control group was 2%, and the high positive expression rate was 0; the positive expression rate of the S100A11 protein in the liver cancer tissue is 98 percent, and the high positive expression rate is 52 percent; the S100A11 protein is obviously increased in liver cancer tissues, and compared with remote normal tissues, the expression level difference of the S100A11 protein has statistical significance.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (2)
1. A kit for early detection and diagnosis of liver cancer metastasis is characterized by comprising a mouse anti-human S100A11 monoclonal antibody, a confining liquid, a working solution, an antibody diluent, a goat anti-human secondary antibody, a DAB developing solution, a sodium citrate buffer solution with the concentration of 0.01mol/L, a hematoxylin staining solution, a neutral resin, 70% ethanol, 75% ethanol, 80% ethanol, 95% ethanol, absolute ethanol, xylene and PBS;
the confining liquid is a 5% bovine serum albumin solution prepared by using phosphate buffer PBS as a solvent; the working solution is prepared by diluting 30% H with PBS solution2O2Solution dilution to 3% H2O2A solution; the antibody diluent comprises 0.1% (V/V) Tween-20 phosphate and 0.1MPBS solution with pH7.4.
2. The use method of the kit for the early detection and diagnosis of liver cancer metastasis according to claim 1, comprising the steps of:
1) taking liver tissues of a person to be detected to carry out paraffin embedding slicing, placing the liver tissues in a 60 ℃ oven for 15min, and dewaxing the paraffin slices to water to obtain dewaxed slices;
2) placing the dewaxed slices in xylene for 10min, replacing xylene and soaking for 10min, then soaking in absolute ethanol for 5min, soaking in 95% ethanol for 5min, soaking in 80% ethanol for 5min, soaking in 75% ethanol for 5min, soaking in 70% ethanol for 5min, soaking in PBS for 5min, then repeatedly soaking in PBS for 3 times, and obtaining slices after primary treatment;
3) placing the slice after the preliminary treatment in a working solution for incubation for 20min at room temperature to eliminate the activity of endogenous peroxidase to obtain the slice after enzyme deactivation treatment;
4) washing the slices after enzyme deactivation with distilled water, then soaking for 5min with PBS, and repeatedly soaking for 3 times with PBS replaced to obtain washed slices;
5) placing the cleaned slices into a sodium citrate buffer solution with the concentration of 0.01mol/L, placing the slices into a microwave oven to be heated at the temperature of 90-99 ℃, then placing the slices at room temperature for 11-15min, and repeating the steps for 3 times to obtain the slices after antigen retrieval;
6) placing the sections subjected to antigen restoration in a sealing solution, incubating at room temperature for 20-30 min, removing the sealing solution, dropwise adding a mouse anti-human S100A11 monoclonal antibody into the sections, and incubating at 37 ℃ for 1-2 hours to obtain treated sections;
7) soaking the treated slices in PBS for 5min, and repeatedly soaking for 3 times by replacing PBS;
8) adding a goat anti-human secondary antibody marked by HRP (horse radish peroxidase) dropwise into the processed section, and incubating at 37 ℃ for 60min to obtain a section processed by the secondary antibody;
9) soaking the slices treated by the secondary antibody in PBS for 5min, and repeatedly soaking for 3 times by replacing PBS;
10) dripping a proper amount of working solution into the processed slices, and incubating for 60min at 37 ℃;
11) soaking the processed slices in PBS for 5min, and repeatedly soaking for 3 times by replacing PBS;
12) placing the processed slices in DAB developing solution for developing color at room temperature, observing the developing process of the slices under a microscope, controlling the reaction time to be 5-10 min, and washing the slices with distilled water to obtain the developed slices;
13) dropwise adding hematoxylin staining solution on the slices after the color development, slightly re-staining the slices, then differentiating the slices for 30s by using hydrochloric acid and alcohol, and washing the slices for 10min by using distilled water to obtain the re-stained slices;
14) soaking the slices after the redyeing in 75% ethanol for 5min and 80% ethanol for 5min, soaking in 95% ethanol for 5min and repeating for 3 times, soaking in 100% ethanol for 5min and repeating for 3 times, soaking in xylene for 20min and repeating for 3 times, and sealing the slices with neutral gum;
15) the microscope photographs the mounting film, and a DPController image acquisition system is used for acquiring images; after image acquisition, immunohistochemistry scores were given according to cell staining intensity and positive area.
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| WO2011038763A1 (en) * | 2009-09-30 | 2011-04-07 | Franco Maria Buonaguro | Method for biomolecular detection of human liver diseases compositions and kits used in said method |
| US20160215349A1 (en) * | 2013-09-20 | 2016-07-28 | Jeffrey LORDAN | Cancer biomarker and diagnostic |
| CN107831308A (en) * | 2017-11-22 | 2018-03-23 | 南宁科城汇信息科技有限公司 | Transcript profile and the ImmunohistochemistryMethods Methods of protein science in a kind of liver cancer biological process |
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|---|---|---|---|---|
| WO2011038763A1 (en) * | 2009-09-30 | 2011-04-07 | Franco Maria Buonaguro | Method for biomolecular detection of human liver diseases compositions and kits used in said method |
| US20160215349A1 (en) * | 2013-09-20 | 2016-07-28 | Jeffrey LORDAN | Cancer biomarker and diagnostic |
| CN107831308A (en) * | 2017-11-22 | 2018-03-23 | 南宁科城汇信息科技有限公司 | Transcript profile and the ImmunohistochemistryMethods Methods of protein science in a kind of liver cancer biological process |
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Application publication date: 20201023 |