CN111122876A - Application of CD47 antibody in detection kit for sensitivity of liver cancer radiotherapy - Google Patents
Application of CD47 antibody in detection kit for sensitivity of liver cancer radiotherapy Download PDFInfo
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Abstract
The invention provides application of a CD47 antibody in a detection kit for sensitivity of liver cancer radiotherapy. The invention has the advantages that (a) the marker for predicting the treatment effect of the liver radiotherapy is provided, and the selection of a treatment scheme of a patient is facilitated. (b) The sensitivity of the patient to radiotherapy is analyzed in advance, and ineffective treatment is avoided. (c) The detection method of the peripheral blood CD47 is more convenient and rapid and is more easily accepted by patients.
Description
Technical Field
The invention relates to an application of a CD47 antibody in a detection kit for sensitivity of liver cancer radiotherapy, belonging to the field of biological detection.
Background
Primary liver cancer (hereinafter, referred to as liver cancer) is the cancer with six global morbidity and the third mortality. In addition, the incidence of liver cancer complicated with portal vein cancer emboli is high. Once combined with portal vein emboli, patients often have poor prognosis. Therefore, portal vein thrombosis is an important factor affecting prognosis of liver cancer. In recent years, with the development of science and technology and the improvement of medical level, especially the improvement of operation method and the popularization of multidisciplinary diagnosis and treatment concept, the treatment of liver cancer combined with portal vein cancer suppository has been advanced greatly. In 2004, the group proposed the classification of portal vein cancer emboli for the first time in China aiming at different degrees of severity of portal vein cancer emboli invading the portal vein. The method comprises the following specific steps: depending on the extent of development of the cancer embolus (i.e. invasion of different portal vein sites), cancer emboli are classified as type I-IV: the microvascular cancer embolus is stage I0, and the type I is the one with cancer embolus affecting more than two levels of portal vein branches; type II in case of involvement of the first portal vein branch; type III in case of involvement of portal trunk; those affecting the superior or inferior mesenteric vein are type IV.
At present, the treatment scheme for removing liver cancer accompanied by type I and type II portal vein cancer embolus is accepted by people, and the operation is the scheme selected above. However, there is still controversy for the treatment of type III, IV portal vein cancer emboli. Therefore, for controversial treatment of III and IV type portal cancer emboli, prospective clinical research is designed, one group is subjected to surgical treatment, the other group is subjected to preoperative small-dose cancer emboli radiotherapy, the thin-layer CT of the liver is rechecked after 3 weeks, the volume ranges of tumors and cancer emboli are determined by a digital three-dimensional reconstruction technology, the radiotherapy effect and the auxiliary surgical positioning are determined, and the descending surgical treatment is performed in parallel. Finally, the prognosis of two groups of patients is compared, and the curative effect of the group of patients treated by III and IV cancer embolus radiotherapy and the phase-down operation is obviously better than that of the group treated by simple operation. However, the curative effect of radiotherapy for patients with liver cancer is significantly different, so that a technology capable of accurately providing guidance for the treatment of clinical type III and IV cancer emboli patients is urgently needed.
Disclosure of Invention
In order to provide guidance for treating clinical type III and type IV cancer embolus patients more accurately, the expression of the serum and postoperative tissues of radiotherapy patients is detected, and the correlation between the CD47 and the result of liver cancer radiotherapy is found. Therefore, the application of the CD47 antibody in the detection kit is constructed in the invention, so as to help judge whether the patient is suitable for radiotherapy treatment in advance, and provide diagnosis help for better improving the prognosis of the patient in clinic.
The invention adopts the following technical scheme:
the CD47 antibody is applied to a detection kit for the sensitivity of liver cancer radiotherapy.
Further, the application of the invention comprises the following steps:
step one, manufacturing a tissue slice,
step two, detecting the expression level of CD47 in the tissue section,
and step three, grading and judging.
Further, the application of the invention also has the following characteristics:
in the first step, a tissue section is prepared by adopting a paraffin embedding method, and the method comprises the following steps:
(i) sampling for 30min, and putting into a fixing solution, wherein the dosage of the fixing solution is about 20 times of that of the tissue block, the fixing time is 24h, and the fixing solution comprises 10% formalin, 4% paraformaldehyde or 10% phosphoric acid buffered formalin;
(ii) after the fixed sample is washed by running water (4-6 h), ethanol with different concentrations is used for dehydration treatment, and the ethanol concentration is as follows: 30% → 50% → 70% → 80% → 90% → 95% → 100% (I) → 100% (II), the volume of ethanol in each concentration being about 50 times that of the tissue mass, and the total time period being about 2 to 3 hours;
(iii) placing the tissue block in a transparent agent with a ratio of xylene to absolute ethyl alcohol of 1:1 for 1h, and then placing the tissue block in xylene for transparency for 20 min;
(iv) putting the transparent tissue block into normal paraffin in a dissolved state for 1h, putting the material containing paraffin, putting the tissue block after being soaked in the paraffin into a container (arranged at a position in the paraffin) containing paraffin liquid, and quickly putting the tissue block into cold water for cooling after the surface layer of the paraffin liquid is solidified to prepare the paraffin block containing the tissue block;
(v) the embedded wax block is trimmed into a regular quadrangular frustum pyramid by a blade, the bottom of the regular quadrangular frustum pyramid is quickly attached to a small wood block by a small amount of hot wax liquid, the small wood block is sliced by a rotary slicer, the slice thickness is 4-7 mu m, a wax tape which is connected with one another is cut, the wax tape is lightly held by a writing brush and is put on paper, and the flattened wax sheet is firmly attached to a glass slide by an adhesive.
Further, the application of the invention also has the following characteristics:
in the first step, a freezing method is adopted to prepare a tissue slice, and the method comprises the following steps:
(i) placing the tissue block on a supporter, dripping embedding medium, and placing on a freezing table for freezing;
(ii) and clamping the frozen tissue block on a holder of a freezing microtome, trimming and slicing, wherein the thickness of the slice is 5-10 mu m, and flatly adhering the sliced slice on a glass slide.
Further, the application of the invention also has the following characteristics:
in the second step, the detection of the expression level of CD47 in the tissue section comprises the following steps:
(1) dewaxing and hydrating, namely taking out the tissue before dewaxing, putting the tissue in a 65 ℃ oven for 1h, then soaking the tissue in dimethylbenzene for 2 times, 10min each time, then sequentially adding 100% absolute ethyl alcohol, 90% absolute ethyl alcohol, 80% absolute ethyl alcohol and 70% absolute ethyl alcohol for 5min respectively, and finally putting the tissue in distilled water for 5 min;
(2) antigen retrieval: heating sodium citrate buffer solution in water bath to about 95 deg.C, and repairing with paraffin section for 15-30 min;
(3) after cooling at room temperature, washing with PBS for 3 times, then incubating with 3% H2O2 at room temperature for 30min, and washing with PBS for 3 times, each time for 5 min;
(4) sealing with 5% goat serum, incubating at room temperature for 30min,
(5) dripping primary antibody working solution on the slices, placing the slices in a wet box at 4 ℃ for incubation overnight,
(6) washing with PBS for 3 times, each for 5min, wiping dry the slices, adding secondary antibody working solution dropwise, placing in a wet box, and incubating for 2 hours at room temperature;
(7) washing with PBS for 3 times, each for 5min, dripping DAB color developing solution, observing color change under microscope, and washing with distilled water to stop reaction;
(8) HE counterdyeing, dehydrating, and sealing with neutral resin.
Further, the application of the invention also has the following characteristics:
in the third step, the detection results are divided into: negative (-) expression, weak positive (+) expression and strong positive (+) expression, the strong positive expression is judged to be not suitable for the treatment by radiotherapy.
Further, the application of the invention can also comprise the following steps:
step one, preparing a plate:
step two, a determination step;
and step three, grading and judging.
Further, the application of the present invention may also have the following features:
the first step comprises the following steps:
(1) the capture antibody was diluted to working concentration in PBS without carrier protein, 96-well microplates were immediately coated with 100 μ Ι _ of diluted capture antibody per well, sealed plates and incubated overnight at room temperature,
(2) sucking out each well and washing with washing buffer, repeating the process twice for 3 times, removing all the remaining washing liquid by sucking or flipping the plate and dipping it on a clean paper towel after the last washing,
(3) add 300. mu.L of reagent diluent to each well to block the plate, incubate at room temperature for at least 1 hour,
(4) the suction/irrigation step in step 2 is repeated.
Further, the application of the present invention may also have the following features: the second step comprises the following steps:
(1) add 100. mu.L of sample or standard per well, cover the wells and incubate at room temperature for 2 hours,
(2) the suction/irrigation is repeated and the suction/irrigation is repeated,
(3) add 100. mu.L of detection antibody to each well, cover the wells and incubate at room temperature for 2 hours,
(4) the suction/irrigation is repeated and the suction/irrigation is repeated,
(5) add 100. mu.L of streptavidin-HRP working dilution to each well, cover the wells and incubate at room temperature for 20min,
(6) the suction/irrigation operation is repeated and the suction/irrigation operation is repeated,
(7) to each well 100. mu.L of substrate solution was added, incubated at room temperature for 20 minutes,
(8) to each well was added 50 μ L of stop solution,
(9) the absorbance of each well was measured using a microplate reader set at 450 nm.
Further, the application of the present invention may also have the following features: in step three, according to the dyeing intensity: 0 ═ no staining, 1 ═ weak staining, 2 ═ moderate staining and 3 ═ strong staining, and the percentage of stained cells: 0-0%, 1-24%, 2-25-49%, 3-50-74% and 4-75-100%, and the immunoreaction score was determined by multiplying the intensity score by the percentage of stained cells. As a result, the scores of 9 ranks were 0, 1, 2, 3, 4, 6, 8, 9 and 12, respectively, with low expression at a score of 4 or less, high expression at a score of 5 or more indicating unsuitability for radiotherapy.
Advantageous effects of the invention
The application of the CD47 antibody in the detection kit for the sensitivity of the liver cancer radiotherapy has the advantages that the CD47 kit is simple and convenient, and the expression of CD47 is stable, so that a clinician can be well helped to judge the curative effect of the radiotherapy of a liver cancer patient.
The main advantages of the invention include:
(a) markers predictive of therapeutic efficacy for liver radiotherapy are provided, facilitating selection of a patient treatment regimen.
(b) The sensitivity of the patient to radiotherapy is analyzed in advance, and ineffective treatment is avoided.
(c) The detection method of the peripheral blood CD47 is more convenient and rapid and is more easily accepted by patients.
Drawings
FIG. 1A is a negative result of immunohistochemical detection of CD47 expression levels;
FIG. 1B is a weak positive result of immunohistochemistry for detecting the expression level of CD 47;
FIG. 1C is a strong positive result of immunohistochemical detection of CD47 expression levels;
figure 1D survival curves for two groups of patients.
Detailed Description
The technical solution of the present invention is further described below with reference to specific embodiments.
The invention discovers that the concentration or the level of CD47 in the tumor tissue and the peripheral blood of the liver cancer patient has obvious correlation with the sensitivity of the liver cancer patient to radiotherapy for the first time. Therefore, the CD47 antibody can be used in diagnostic reagent or diagnostic kit for judging the treatment effect of liver cancer patients.
CD47 protein and gene sequence:
human gene CD47(CD47 molecule), GenBank accession NC-000003, full length 51107bp, open reading frame containing 972 nucleotides, encoding 323 amino acid residues.
CD47 is present in tumor tissue and/or blood.
< example one >
The present example provides the use of a CD47 antibody in a kit for determining the effectiveness of a radiation therapy and/or determining whether a tumor patient is eligible for a radiation therapy. The CD47 antibody of the present invention may be monoclonal antibody, polyclonal antibody, or their effective fragments, or may be obtained by other genetic engineering means.
In the present embodiment, an immunohistochemical detection kit is exemplified.
In the application of the present embodiment, the method for producing a paraffin section may include the steps of:
(i) sampling, wherein the length of the tissue sample is 3-6 mm, the width is 3-6 mm, and the thickness is 1-3 mm. After sampling, the tissue blocks are put into a fixing solution within 30min, the dosage of the fixing solution is about 20 times of that of the tissue blocks, the fixing time is 24h, and the fixing solution comprises 10% formalin, 4% paraformaldehyde or 10% phosphate buffered formalin;
(ii) after the fixed sample is washed by running water (4-6 h), ethanol with different concentrations is used for dehydration treatment, and the ethanol concentration is as follows: 30% → 50% → 70% → 80% → 90% → 95% → 100% (I) → 100% (II), the volume of ethanol in each concentration being about 50 times that of the tissue mass, and the total time period being about 2 to 3 hours;
(iii) placing the tissue block in a clearing agent with a ratio of xylene to absolute ethyl alcohol of 1:1 for 1h, and then placing in xylene for clearing for 20 min;
(iv) putting the transparent tissue block into normal paraffin for 1h, putting the tissue block after being soaked in paraffin into a container filled with paraffin liquid, and quickly putting the tissue block into cold water for cooling after the surface layer of the paraffin liquid is solidified to prepare a paraffin block containing the tissue block;
(v) the embedded wax block is trimmed into a regular quadrangular frustum pyramid by a blade, the bottom of the regular quadrangular frustum pyramid is attached to a small wood block by a small amount of hot wax liquid, the small wood block is sliced by a rotary slicer, the slice thickness is about 4-7 mu m, the small wood block is lightly held on paper by a writing brush, and the flattened wax sheet is firmly attached to a glass slide by an adhesive.
In other embodiments, the tissue sample may be prepared as a cryo-section for testing.
The preparation of the frozen section comprises the following steps:
(i) placing the tissue block on a supporter, dripping embedding medium, and freezing on a freezing table;
(ii) and clamping the frozen tissue block on a holder of a freezing microtome, trimming and slicing, wherein the thickness of the slice is 5-10 mu m, and flatly adhering the sliced slice on a glass slide.
Detection of CD47 expression levels in tissue sections
(1) Dewaxing and hydrating, namely taking out the tissue before dewaxing, putting the tissue in a 65 ℃ oven for 1h, then soaking the tissue in dimethylbenzene for 2 times, 10min each time, then sequentially adding 100% absolute ethyl alcohol, 90% absolute ethyl alcohol, 80% absolute ethyl alcohol and 70% absolute ethyl alcohol for 5min respectively, and finally putting the tissue in distilled water for 5 min;
(2) antigen retrieval: heating sodium citrate buffer solution in water bath to about 95 deg.C, and repairing with paraffin section for 15-30 min;
(3) after cooling at room temperature, washing with PBS 3 times, incubating with 3% H2O2 at room temperature for 30min to eliminate interference of endogenous peroxidase, washing with PBS 3 times for 5min each
(4) 5% goat serum (PBS diluted)) and incubated at room temperature for 30min, gently shaking off the residue on the sections;
(5) dripping a primary antibody working solution (proteintech 20305-1-AP) prepared in a certain proportion on the section, and incubating overnight in a wet box at 4 DEG C
(6) Washing with PBS for 3 times, each time for 5min, wiping dry the slices, adding secondary antibody working solution (proteintech SA00004-2) dropwise, placing in a wet box, and incubating for 2 hours at room temperature;
(7) washing with PBS for 3 times, each for 5min, dripping DAB color developing solution, observing color change under microscope, and washing with distilled water to stop reaction;
(8) HE counterdyeing, dehydrating, and sealing with neutral resin;
(i) tissue slice pretreatment
(ii) Washing the section with PBS buffer solution, blocking with serum (the serum concentration is recommended according to the instruction), dropping CD47 antibody for staining (Proteintetech 20305-1-AP) (the volume ratio of the antibody to the PBS is 1:200 to 1:500), and incubating at 37 ℃ for 1-2 h, or incubating at 4 ℃ overnight;
(iii) washing with PBS buffer solution, wiping off PBS buffer solution around the tissue, dripping biotin-derived secondary antibody, and incubating at 37 deg.C for 30 min;
(iv) washing with PBS buffer solution, adding three antibodies (SAB complex), and incubating at 37 deg.C for 40 min;
(v) after the color development of Diaminobenzidine (DAB), the color development reaction is stopped by flushing with running water, and the flushing with running water is carried out after the hematoxylin counterstaining;
(vi) after the gradient ethanol solution is dehydrated and the xylene is transparent, the mixture is sealed by neutral resin and observed by a microscope.
In other embodiments, in step (i), the tissue section is a frozen section, and the pretreatment step is to fix the section with cold acetone at 4 ℃ for 10-20 min.
Judging a dyeing result:
the staining results were divided into three categories: negative (-) expression, weak positive (+) expression, and strong positive (+) expression. The detection results were divided into a low expression group and a high expression group, which are expressed as + and + +, respectively. The high expression panel suggests that the patient may have poor radiotherapy efficacy.
< example two >
Application of CD47 antibody in detection kit for sensitivity of liver cancer radiotherapy
The embodiment provides a kit for detecting sensitivity of radiotherapy of liver cancer, which contains anti-CD 47 immunoglobulin or immunoconjugate, or active fragment thereof.
The detection process is as follows:
preparation of the plate:
1. the capture antibody was diluted to working concentration in PBS without carrier protein. The 96-well microplate was immediately coated with 100. mu.L of diluted capture antibody per well. Plates were sealed and incubated overnight at room temperature.
2. Each well was aspirated and washed with Wash Buffer, and the procedure was repeated twice for a total of 3 washes. Cleaning was performed by filling each well with a spray bottle, manifold dispenser or automatic cleaner using a rinse bottle (400 μ L). Thorough removal of the liquid in each step is critical to good performance.
3. The plate was blocked by adding 300 μ L of reagent diluent to each well. Incubate at room temperature for at least 1 hour.
4. The suction/irrigation step in step 2 is repeated.
The determination step comprises:
1. each well was filled with 100. mu.L of peripheral blood sample or standard in a reagent diluent or appropriate diluent. Covered with tape and incubated at room temperature for 2 hours.
2. Suction and flushing are repeated as per step 2 of plate preparation.
3. To each well 100 μ L of detection antibody diluted with reagent diluent was added, capped and incubated at room temperature for 2 hours.
4. The pumping/flushing is repeated according to step 2 of the plate preparation.
5. To each well 100 μ L of streptavidin-HRP working dilution was added. Plates were covered and incubated at room temperature for 20 minutes. Avoiding placing the board under direct light.
6. The suction and irrigation operations in step 2 are repeated.
7. To each well 100. mu.L of substrate solution was added. Incubate at room temperature for 20 minutes. Avoiding placing the board under direct light.
8. To each well 50 μ L of stop solution was added. Gently tap the dish to ensure adequate mixing.
9. The optical density of each well was immediately determined using a microplate reader set at 450 nm. If wavelength correction is possible, 540nm or 570nm is set. If no wavelength correction is possible, either the 540nm or 570nm reading is subtracted from the 450nm reading. This subtraction will correct for optical defects in the plate.
The radiotherapy effect detection kit of the invention has completed 80 experiments.
After the patients are treated by radiotherapy, the detection kit disclosed by the invention is used for detecting the objects of the CD47 high-expression group, and the curative effect of the radiotherapy and the survival time of the liver cancer are obviously shorter than those of the patients of the CD47 low-expression group. The expression level is determined by the intensity and area. The percentage of stained cells (0 ═ 0%, 1 ═ 24%, 2 ═ 25 to 49%, 3 ═ 50 to 74% and 4 ═ 75 to 100%) was determined from the intensity of staining (0 ═ weak staining, 1 ═ weak staining, 2 ═ moderate staining and 3 ═ strong staining). The final immune response score was determined by multiplying the intensity score by the score for the percentage of stained cells. As a result, scores of 0, 1, 2, 3, 4, 6, 8, 9 and 12 were respectively given to 9 ranks, and then the scores were analyzed. The expression was judged to be low in score 4 or less. High expression was judged by score 5 or more.
< example three >
Verification of application effect of CD47 in kit
The effectiveness of the technical scheme of the invention is verified by taking a tissue section of a patient with tumor resection as an example.
In this example, the patients with liver cancer were all patients who had undergone surgery before and after radiotherapy. Liver cancer tumor tissue surgically removed from the patient was examined for CD47 expression level, and the procedure was as described in example one.
Judgment of result of detecting CD47 expression level by immunohistochemical method
FIG. 1 shows the results of immunohistochemical detection of the expression level of CD 47. Wherein, 1A is negative (-) expression, 1B is weak positive (+) expression, and 1C is strong positive (+) expression. The detection results were divided into a low expression group and a high expression group, which are expressed as + and + +, respectively.
As shown in fig. 1D, liver cancer survival was significantly longer in subjects with low expression of CD47 than in subjects with high expression of CD47 (P <0.05, results were statistically significant).
The above examples show that before radiotherapy treatment of a liver cancer patient, the application of the present invention is used for detection, so that a more accurate treatment scheme can be adopted for the patient, the treatment effect is improved, ineffective treatment is prevented, and the life cycle of the patient is prolonged.
Claims (10)
- Application of a CD47 antibody in a kit for detecting sensitivity of liver cancer radiotherapy.
- 2. Use according to claim 1, characterized in that it comprises the following steps:step one, manufacturing a tissue slice,step two, detecting the expression level of CD47 in the tissue section,and step three, grading and judging.
- 3. Use according to claim 2, characterized in that:in the first step, a tissue section is prepared by adopting a paraffin embedding method, and the method comprises the following steps:(i) sampling for 30min, and putting into a fixing solution, wherein the dosage of the fixing solution is about 20 times of that of the tissue block, the fixing time is 24h, and the fixing solution comprises 10% formalin, 4% paraformaldehyde or 10% phosphoric acid buffered formalin;(ii) after the fixed sample is washed by running water (4-6 h), ethanol with different concentrations is used for dehydration treatment, and the ethanol concentration is as follows: 30% → 50% → 70% → 80% → 90% → 95% → 100% (I) → 100% (II), the volume of ethanol in each concentration being about 50 times that of the tissue mass, and the total time period being about 2 to 3 hours;(iii) placing the tissue block in a transparent agent with a ratio of xylene to absolute ethyl alcohol of 1:1 for 1h, and then placing the tissue block in xylene for transparency for 20 min;(iv) putting the transparent tissue block into normal paraffin in a dissolved state for 1h, putting the material containing paraffin, putting the tissue block after being soaked in the paraffin into a container containing paraffin liquid, and quickly putting the tissue block into cold water for cooling after the surface layer of the paraffin liquid is solidified to prepare the paraffin block containing the tissue block;(v) the embedded wax block is trimmed into a regular quadrangular frustum pyramid by a blade, the bottom of the regular quadrangular frustum pyramid is quickly attached to a small wood block by a small amount of hot wax liquid, the small wood block is sliced by a rotary slicer, the slice thickness is 4-7 mu m, a wax tape which is connected with one another is cut, the wax tape is lightly held by a writing brush and is put on paper, and the flattened wax sheet is firmly attached to a glass slide by an adhesive.
- 4. Use according to claim 2, characterized in that:in the first step, a freezing method is adopted to prepare a tissue slice, and the method comprises the following steps:(i) placing the tissue block on a supporter, dripping embedding medium, and placing on a freezing table for freezing;(ii) and clamping the frozen tissue block on a holder of a freezing microtome, trimming and slicing, wherein the thickness of the slice is 5-10 mu m, and flatly adhering the sliced slice on a glass slide.
- 5. The use of claim 1, wherein:in the second step, the detection of the expression level of CD47 in the tissue section comprises the following steps:(1) dewaxing and hydrating, namely taking out the tissue before dewaxing, putting the tissue in a 65 ℃ oven for 1h, then soaking the tissue in dimethylbenzene for 2 times, 10min each time, then sequentially adding 100% absolute ethyl alcohol, 90% absolute ethyl alcohol, 80% absolute ethyl alcohol and 70% absolute ethyl alcohol for 5min respectively, and finally putting the tissue in distilled water for 5 min;(2) antigen retrieval: heating sodium citrate buffer solution in water bath to about 95 deg.C, and repairing with paraffin section for 15-30 min;(3) after cooling at room temperature, washing with PBS 3 times, incubating with 3% H2O2 at room temperature for 30min, washing with PBS 3 times, each for 5min(4) Sealing with 5% goat serum, incubating at room temperature for 30min,(5) dripping primary antibody working solution on the slices, placing the slices in a wet box at 4 ℃ for incubation overnight,(6) washing with PBS for 3 times, each for 5min, wiping dry the slices, adding secondary antibody working solution dropwise, placing in a wet box, and incubating for 2 hours at room temperature;(7) washing with PBS for 3 times, each for 5min, dripping DAB color developing solution, observing color change under microscope, and washing with distilled water to stop reaction;(8) HE counterdyeing, dehydrating, and sealing with neutral resin.
- 6. The use of claim 5, wherein:in the third step, the detection results are divided into: negative (-) expression, weak positive (+) expression and strong positive (+) expression, the strong positive expression is judged to be not suitable for the treatment by radiotherapy.
- 7. Use according to claim 1, characterized in that it comprises the following steps:step one, preparing a plate:step two, a determination step;and step three, grading and judging.
- 8. The use of claim 7, wherein:the first step comprises the following steps:(1) the capture antibody was diluted to working concentration in PBS without carrier protein, 96-well microplates were immediately coated with 100 μ Ι _ of diluted capture antibody per well, sealed plates and incubated overnight at room temperature,(2) sucking out each well and washing with washing buffer, repeating the process twice for 3 times, removing all the remaining washing liquid by sucking or flipping the plate and dipping it on a clean paper towel after the last washing,(3) add 300. mu.L of reagent diluent to each well to block the plate, incubate at room temperature for at least 1 hour,(4) the suction/irrigation step in step 2 is repeated.
- 9. The use of claim 7, wherein:the second step comprises the following steps:(1) add 100. mu.L of sample or standard per well, cover the wells and incubate at room temperature for 2 hours,(2) the suction/irrigation is repeated and the suction/irrigation is repeated,(3) add 100. mu.L of detection antibody to each well, cover the wells and incubate at room temperature for 2 hours,(4) the suction/irrigation is repeated and the suction/irrigation is repeated,(5) add 100. mu.L of streptavidin-HRP working dilution to each well, cover the wells and incubate at room temperature for 20min,(6) the suction/irrigation operation is repeated and the suction/irrigation operation is repeated,(7) to each well 100. mu.L of substrate solution was added, incubated at room temperature for 20 minutes,(8) to each well was added 50 μ L of stop solution,(9) the absorbance of each well was measured using a microplate reader set at 450 nm.
- 10. The use of claim 7, wherein:in step three, according to the dyeing intensity: 0 ═ no staining, 1 ═ weak staining, 2 ═ moderate staining and 3 ═ strong staining, and the percentage of stained cells: 0-0%, 1-24%, 2-25-49%, 3-50-74% and 4-75-100%, the immunoreaction score was determined by multiplying the intensity score by the percentage of stained cells, the scores for the 9 grades were 0, 1, 2, 3, 4, 6, 8, 9 and 12, respectively, 4 and below was low expression, 5 and above was high expression, and high expression indicated that radiotherapy was not suitable.
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