CN111289748A - Application of ErbB3 protein in preparation of bladder cancer noninvasive diagnosis product - Google Patents
Application of ErbB3 protein in preparation of bladder cancer noninvasive diagnosis product Download PDFInfo
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- CN111289748A CN111289748A CN201811499065.XA CN201811499065A CN111289748A CN 111289748 A CN111289748 A CN 111289748A CN 201811499065 A CN201811499065 A CN 201811499065A CN 111289748 A CN111289748 A CN 111289748A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—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
- G01N33/57488—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 involving compounds identifable in body fluids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
Abstract
The invention discloses an application of ErbB3 protein in preparing a bladder urothelial cancer noninvasive diagnosis product, which comprises the following steps: the invention has reasonable structure and safe and convenient use, and can diagnose whether the patient has bladder urothelial carcinoma or not by detecting the content of the ErbB3 protein in the urine of the patient; the ErbB3 protein is used for distinguishing a diagnosis threshold value of bladder high-grade and low-grade urothelial cancer, and tumor malignancy grade information can be provided by detecting the content of ErbB3 in urine of a patient, so that the ErbB3 protein has a certain reference value for preoperative evaluation and postoperative follow-up; thirdly, the muscle layer infiltration type bladder urothelial cancer is easy to transfer far, and the diagnosis threshold value of the ErbB3 protein in the urine of a patient to the muscle layer infiltration type tumor is determined by utilizing the noninvasive diagnosis product; has great significance for more accurate clinical diagnosis and treatment strategy formulation.
Description
Technical Field
The invention relates to the technical field of bladder cancer diagnosis, in particular to application of ErbB3 protein in preparation of a bladder urothelial cancer noninvasive diagnosis product.
Background
The bladder cancer is a malignant tumor which occurs in the bladder, the tissue type of the malignant tumor belongs to the urothelial cancer mostly, the malignant tumor can be divided into high-grade urothelial cancer and low-grade urothelial cancer according to different tumor cell forms and biological behaviors, the malignant tumor is divided into invasive urothelial cancer and non-invasive urothelial cancer according to different infiltration degrees, the incidence rate of the bladder urothelial cancer is in the rising trend at present, the tumor is the first tumor of the incidence rate of the urogenital system tumor, therefore, the diagnosis and treatment effects on the bladder tumor are improved, and the diagnosis and treatment effects on the bladder tumor are of great significance to national health;
the clinical manifestations of bladder cancer patients are mostly painless, intermittent and whole-course naked eye hematuria and under-the-mirror hematuria, few patients can firstly have bladder irritation symptoms such as frequent micturition, urgent micturition and painful urination, some patients are intravesical tumors discovered by imaging accidentally during physical examination, the treatment effect of bladder cancer is closely related to whether tumors can be discovered early, the prognosis of patients diagnosed early is good, the survival rate of 5 years after operation can reach more than 90%, the pathological biopsy of specimens obtained by urethral cystoscopy is the current gold standard for diagnosing bladder tumors, but the method has invasiveness, is not easy to popularize for population screening, urine exfoliative cytology examination is also a common method, but the method has low sensitivity and is easily affected by urinary tract infection, stones and the like, so that it is necessary to discover a high-efficiency diagnosis marker from urine to diagnose and treat bladder tumors, the research finds that the detection of the content of the ErbB3 protein in urine is expected to help the diagnosis and disease follow-up of the urinary bladder urothelial cancer population.
Disclosure of Invention
The invention provides application of ErbB3 protein in preparation of bladder cancer noninvasive diagnosis products, which can effectively solve the problems that transurethral cystoscopy specimen taking and pathological biopsy provided in the background technology are the current gold standard for diagnosing bladder tumors, but the method is invasive, is not easy to popularize for people screening, and urine cast-off cytology examination is a common method, but the method is low in sensitivity and is easily influenced by urinary tract infection, stones and the like.
In order to achieve the purpose, the invention provides the following technical scheme: the application of the ErbB3 protein in preparing a bladder cancer noninvasive diagnosis product comprises the following steps:
s1, acquiring medical history and examining physique of a patient suspected to suffer from bladder urothelial cancer;
s2, preparing the patient meeting the diagnosis condition before diagnosis;
s3, preparing materials required for diagnosis;
s4, after the ELISA plate coated with the antibody is balanced to the room temperature, adding 100 mu L of prepared urine samples into corresponding holes;
s5, adding a biotin-labeled anti-ErbB 3 antibody into the ELISA plate coated with the antibody;
s6, adding diluted HRP-streptavidin into the ELISA plate coated with the antibody;
s7, sequentially adding TMB color development solution and stop solution into the ELISA plate coated with the antibody, and reading by using an ELISA reader at 450 nm;
s8, calculating a concentration value by adopting sigmaplot12.0 software;
s9, comparing the calculated ErbB3 protein concentration value with the ErbB3 protein concentration value of a healthy population, and diagnosing whether the patient has bladder urothelial cancer.
In step S1, the patient who is diagnosed with bladder urothelial cancer needs to satisfy any preliminary diagnosis suspected bladder urothelial cancer, and has not been treated with anti-tumor therapy such as chemotherapy, radiotherapy or surgery; the patient has not suffered from the related diseases which can have the influence on the urine protein;
in step S2, after confirming that the patient satisfies all the conditions in S1, morning urine of the next day after patient admission is taken, centrifuged at 4500r/min for 10 minutes at 4 ℃ immediately, and at least 2ml of supernatant is stored in a refrigerator at-80 ℃ for later use.
In step S3, the materials required before diagnosis include: the kit comprises an ELISA plate coated with anti-HumanErbB3 antibody, concentrated 20 Xwashing liquid, deionized water, a plate washing machine, biotin-labeled anti-ErbB 3 antibody, concentrated 200 XHRP-streptavidin, TMB color development liquid, stop solution and a 450nm enzyme labeling instrument; diluting the concentrated washing solution to 20 times by using deionized water for later use; centrifuging the concentrated HRP-streptavidin, and then diluting the HRP-streptavidin to 200 times by using deionized water for later use; .
In the step S4, after the antibody-coated ELISA plate is balanced to room temperature, 100 μ L of the prepared standard and the patient urine sample are added to the corresponding wells, the whole plate strip is sealed by a sealing plate film, and the plate strip is incubated overnight at 4 ℃.
In the step S5, the prepared 1x washing liquid is added to a plate washing machine, the plate washing machine is used for washing the plate strips for 5 times, 300 mu Llx washing liquid is added into each hole, after the plate washing machine is washed clean, 100 mu L of the prepared biotin labeled anti-ErbB 3 antibody is added into each hole of the ELISA plate coated with the antibody, and the incubation is carried out for 1h at room temperature.
In the step S6, the prepared 1x washing liquor is added to a plate washing machine, the plate washing machine is used for washing the lath for 5 times, 300 mu Llx washing liquor is added into each hole, after the plate washing machine is washed clean, 100 mu L of prepared HRP-streptavidin is added into each hole of the ELISA plate coated with the antibody, and the incubation is carried out for 45min at room temperature.
In the step S7, adding the prepared 1x washing liquor to a plate washing machine, washing the plate strips for 5 times by using the plate washing machine, adding 300 mu Llx washing liquor into each hole, after the plate washing machine is cleaned, adding 100 mu LTMB developing solution into each hole of the antibody-coated ELISA plate, incubating for 30min at room temperature in a dark place, adding 50 mu L stop solution into each hole of the antibody-coated ELISA plate, and immediately reading at 450nm of an ELISA reader.
In step S8, the ErbB3 protein concentration value of the urine specimen was calculated using sigmaplot12.0 software.
In step S9, the finally obtained value of the ErbB3 protein concentration in the morning urine of the patient is compared with the value of the ErbB3 protein concentration in the urine of healthy people, and a diagnosis is made as to whether the patient has bladder cancer.
Compared with the prior art, the invention has the beneficial effects that: the invention has scientific and reasonable structure and safe and convenient use, and can diagnose whether the patient has bladder urothelial carcinoma or not by detecting the content of the ErbB3 protein in the urine of the patient; the diagnosis values of the ErbB3 protein for further distinguishing the bladder high-grade and low-grade urothelial cancer and the non-invasive urothelial cancer are utilized, and the detection results of corresponding specimens are analyzed, so that the clinical judgment of the tumor grade is facilitated, and whether the risk of bladder wall muscle layer invasion exists is known, thereby having great significance for further making clinical diagnosis and treatment strategies. In addition, by carrying out Spearman correlation analysis on the tumor diameter and the concentration of the ErbB3 protein in urine, the content of the ErbB3 in the urine of a bladder cancer patient is in positive correlation with the medium intensity of the maximum diameter of the tumor, but is not correlated with the age and the BMI of the patient, so that the tumor diameter can be preliminarily judged by utilizing the concentration of the ErbB3 in the urine. Finally, immunohistochemical staining is carried out on ErbB3, positive expression of ErbB3 of 80% bladder urothelium patients is found, important effects are possibly played in the tumor development process, and a foundation is laid for further mechanism research in the future.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
In the drawings:
FIG. 1 is a schematic of the diagnostic process of the present invention.
FIG. 2 is a scatter plot showing the correlation between ErbB3 concentration in urine and tumor maximum size in accordance with the present invention.
FIG. 3 is a schematic representation of the bladder cancer diagnosis of the present invention.
FIG. 4 is a graph showing the diagnostic value for differentiating the high-grade urothelial cancer from the low-grade urothelial cancer of the bladder of the present invention.
FIG. 5 is a standard curve of plate No. 1 of the experimental results.
FIG. 6 is a standard curve of the plate No. 2 of the experimental results.
Fig. 7 is a standard curve of plate No. 3 of the experimental results.
FIG. 8 is the data values of the results of the test for bladder cancer diagnosis.
FIG. 9 is a graph showing the judgment of tumor grade in the results of the experiment.
FIG. 10 is a graph of experimental results showing muscle layer invasion data of bladder wall.
FIG. 11 is a graph of experimental results showing muscle layer invasion data of bladder wall.
FIG. 12 is a table comparing the concentration of ErbB3 protein in urine of bladder cancer patients and normal controls.
FIG. 13 is a table comparing the concentration of ErbB3 in urine from bladder cancer patients with different pathological levels.
FIG. 14 is a table comparing the concentration of ErbB3 in urine from patients with muscle-affected bladder cancer.
FIG. 15 is a table comparing the concentration of ErbB3 in urine from patients with bladder cancer of different sex. Figure 16 is a table comparing the concentration of ErbB3 in urine from patients with diabetic versus normoglycemic bladder cancer as a result of the experiment.
FIG. 17 is a chart comparing the results of the experiments comparing the concentration of ErbB3 in urine from patients with hypertensive bladder cancer.
Figure 18 is a table correlating the concentration of ErbB3 in urine with tumor maximum size, BMI, and age for the results of the experiment.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example (b): as shown in the figure, the invention provides a technical scheme, and the application of ErbB3 protein in preparing bladder cancer noninvasive diagnosis products comprises the following steps:
s1, acquiring medical history and examining physique of a patient suspected to suffer from bladder urothelial cancer;
s2, preparing the patient meeting the diagnosis condition before diagnosis;
s3, preparing materials required for diagnosis;
s4, after the ELISA plate coated with the antibody is balanced to the room temperature, adding 100 mu L of prepared urine samples into corresponding holes;
s5, adding a biotin-labeled anti-ErbB 3 antibody into the ELISA plate coated with the antibody;
s6, adding diluted HRP-streptavidin into the ELISA plate coated with the antibody;
s7, sequentially adding TMB color development solution and stop solution into the ELISA plate coated with the antibody, and reading by using an ELISA reader at 450 nm;
s8, calculating a concentration value by adopting sigmaplot12.0 software;
s9, comparing the calculated ErbB3 protein concentration value with the ErbB3 protein concentration value of a healthy population, and diagnosing whether the patient has bladder urothelial cancer.
In step S1, the patient who is diagnosed with bladder urothelial cancer needs to satisfy any preliminary diagnosis suspected bladder urothelial cancer, and has not been treated with anti-tumor therapy such as chemotherapy, radiotherapy or surgery; the patient has not suffered from the related diseases which can have the influence on the urine protein;
in step S2, after confirming that the patient satisfies all the conditions in S1, morning urine of the next day after patient admission is taken, centrifuged at 4500r/min for 10 minutes at 4 ℃ immediately, and at least 2ml of supernatant is stored in a refrigerator at-80 ℃ for later use.
In step S3, the materials required before diagnosis include: the kit comprises an ELISA plate coated with anti-HumanErbB3 antibody, concentrated 20 Xwashing liquid, deionized water, a plate washing machine, biotin-labeled anti-ErbB 3 antibody, concentrated 200 XHRP-streptavidin, TMB color development liquid, stop solution and a 450nm enzyme labeling instrument; diluting the concentrated washing solution to 20 times by using deionized water for later use; centrifuging the concentrated HRP-streptavidin, and then diluting the HRP-streptavidin to 200 times by using deionized water for later use; .
In the step S4, after the antibody-coated ELISA plate is balanced to room temperature, 100 μ L of the prepared standard and the patient urine sample are added to the corresponding wells, the whole plate strip is sealed by a sealing plate film, and the plate strip is incubated overnight at 4 ℃.
In the step S5, the prepared 1x washing liquid is added to a plate washing machine, the plate washing machine is used for washing the plate strips for 5 times, 300 mu Llx washing liquid is added into each hole, after the plate washing machine is washed clean, 100 mu L of the prepared biotin labeled anti-ErbB 3 antibody is added into each hole of the ELISA plate coated with the antibody, and the incubation is carried out for 1h at room temperature.
In the step S6, the prepared 1x washing liquor is added to a plate washing machine, the plate washing machine is used for washing the lath for 5 times, 300 mu Llx washing liquor is added into each hole, after the plate washing machine is washed clean, 100 mu L of prepared HRP-streptavidin is added into each hole of the ELISA plate coated with the antibody, and the incubation is carried out for 45min at room temperature.
In the step S7, adding the prepared 1x washing liquor to a plate washing machine, washing the plate strips for 5 times by using the plate washing machine, adding 300 mu Llx washing liquor into each hole, after the plate washing machine is cleaned, adding 100 mu LTMB developing solution into each hole of the antibody-coated ELISA plate, incubating for 30min at room temperature in a dark place, adding 50 mu L stop solution into each hole of the antibody-coated ELISA plate, and immediately reading at 450nm of an ELISA reader.
In step S8, the ErbB3 protein concentration value of the urine specimen was calculated using sigmaplot12.0 software.
In step S9, the finally obtained value of the ErbB3 protein concentration in the morning urine of the patient is compared with the value of the ErbB3 protein concentration in the urine of healthy people, and a diagnosis is made as to whether the patient has bladder cancer.
The following are the results of comparative experiments comparing the concentration of ErbB3 in urine of bladder cancer patients and normal controls.
(1) Experimental population
The research standard of urine specimen patients includes the standard of group entry and the standard of exclusion of urine specimen patients, and the standard of group entry of urine specimen patients is:
a. performing surgical treatment such as transurethral cystectomy, partial cystectomy or total cystectomy, and determining the pathological diagnosis after the operation to be a patient with bladder cancer;
b. the patient is treated for the first time and does not carry out anti-tumor treatment such as chemotherapy, radiotherapy or operation;
c. the patient had no other urinary system related diseases;
d. the normal control group was healthy persons who were confirmed by physical examination to be free of urinary and other system-related diseases.
Exclusion criteria were:
a. patients who do not meet the inclusion criteria;
b. the patient is diagnosed again for the recurrence after the urothelial cancer operation;
c. the hospitalization diagnosis is considered as bladder occupation, but the patients are not treated by the operation for various reasons such as economy, worry about operation risks and body conditions unsuitable for the operation and do not obtain pathological biopsy results;
d. the urine is left for standing for too long and is not stored centrifugally and frozen in time;
e. the patient himself or the family members do not agree to join the experimenter.
(2) Sample pretreatment
Morning urine of patients and healthy persons meeting the standard was extracted and immediately centrifuged at 4500r/min at 4 ℃ for 10 minutes, and 7ml of the supernatant was stored in a refrigerator at-80 ℃.
(3) Experimental Material
Reagents, materials and equipment required for the experiment include: the kit comprises an ErBb3 ELISA kit, a Biotek Elx800 ELISA reader, a pipettor, a gun head, a test tube, a centrifuge, a common refrigerator, an ultra-low temperature refrigerator at-80 ℃, filter paper, centrifuge tubes of 500 mu L and 2ml, measuring cylinders of 100 ml and 1L, deionized water and Sigmalplot analysis software;
the components of the ErBb3 ELISA kit are as follows:
ErbB3 Microplate (Item A): the 96-well plate is coated with anti-Human VEGF-A antibody;
wash Buffer Concentrate (20x) (Item B): 25ml of 20 Xconcentrated wash solution;
standards (Item C): recombinant Human ErbB3 standard;
assay Diluent A (Item D): 30 ml for diluting the standard substance and the sample;
assay dilution B (Item E): 15ml of 5x concentrated solution is used for diluting a standard substance and a sample;
detection Antibody ErbB3(Item F): biotin-labeled anti-ErbB 3 antibodies;
HRP-Streptavidin concentrate (Item G): HRP-streptavidin was concentrated at 200 μ L200 ×;
h.TMB One-Step Substrate Reagent (Item H)：12ml3,3’,5,5’-tetramethylbenzidine (TMB)；
stop solution (item I): 8ml of 0.2M sulfuric acid.
(4) Preparation of the experiment
a. The kit and the sample are balanced to room temperature;
b. loading a sample according to the pretreatment result stock solution;
assay dilution B (Item E) was diluted 5-fold with deionized water for use;
d. preparing a standard substance; centrifuging the Item C tubule, adding 400 μ L1 x Assay Diluent B (Item E) to the standard tubule, mixing uniformly to obtain 50ng/ml Standard substance stock solution, sucking 30 μ L of Standard substance stock solution, adding into a centrifugal tube containing 570 μ L1 x Assay dilution B (Item E), mixing uniformly, marking as STD1, preparing 7 1.5 ml centrifugal tubes, adding 400 μ L1 x Assay dilution B (Item E) buffer solution into 7 centrifugal tubes, respectively, marking as STD2, STD3, STD4, STD5, STD6, STD7 and Zero Standard, then, the standard substance is diluted by STD1 gradient of 2500pg/ml, 200 muL of 2500pg/ml standard solution is extracted and added into an STD2 small tube, after uniformly mixing, adding 200ul of the mixture into an STD3 tube for uniformly mixing, and continuing according to the method until STD7 is prepared, wherein Zero Standard is only 400 mu L1 x AssayDiluent A, namely the Standard product is 0 pg/ml;
e. dilution of washing liquor: diluting the concentrated washing solution to 20 times with deionized water, and reserving for later use;
f. detecting an antibody tubule (Item F) by centrifugation, adding 100 mu L of Diluent 1x Assay dilution B (Item E) for full dissolution, slightly hammering up and down, and diluting the antibody tubule (Item F) to 80 times by using 1x Assay dilution B (Item E) Diluent;
g. HRP-streptavidin (Item G) was centrifuged, and then diluted 200-fold with 1 × Assay dilution B (Item E) and used.
(5) Experimental procedure
a. The kit and the sample are balanced to room temperature;
b. after the ELISA plate coated with the antibody is balanced to the room temperature, 100 muL of prepared standard substance and sample are added into the corresponding holes, the whole lath is sealed by a sealing plate film, and the whole lath is incubated overnight at 4 ℃;
c. adding the prepared 1x washing liquor to a plate washing machine, washing the lath for 5 times by using the plate washing machine, and adding 300 mu L of washing liquor into each hole;
d. after washing the plates, adding 100 muL of prepared detection antibody into each hole, and incubating for 1h at room temperature;
e. adding the prepared 1x washing liquor to a plate washing machine, washing the lath for 5 times by using the plate washing machine, and adding 300 mu L of washing liquor into each hole;
f. adding 100 muL of prepared HRP-streptavidin into each hole, and incubating for 45min at room temperature;
g. adding the prepared 1x washing liquor to a plate washing machine, washing the lath for 5 times by using the plate washing machine, and adding 300 mu L of washing liquor into each hole;
h. adding 100 mu of LTMB developing solution into each well, and incubating for 30min at room temperature in a dark place;
i. adding 50 mu L of stop solution into each well, and immediately reading in an enzyme labeling instrument at 450 nm;
j. concentration values were calculated using sigmaplot12.0 software.
The experimental results are shown in the attached drawing
In conclusion: whether the patient has the bladder cancer can be directly diagnosed by detecting the concentration of the ErbB3 protein in the urine of the patient, so that the psychological pressure and the physiological pressure born by the patient in the process of diagnosing the bladder cancer are relieved, and the diagnosis of the bladder cancer is more convenient.
The working principle and the using process of the invention are as follows: the invention has scientific and reasonable structure and safe and convenient use, can diagnose whether a patient has bladder cancer by detecting the content of the ErbB3 protein in the urine of the patient, utilizes the diagnostic value of the ErbB3 protein for further distinguishing high-grade urinary epithelial cancer from low-grade urinary epithelial cancer, finds that the ErbB3 in the urine of the patient is obviously higher than that of the patient with the high-grade urinary epithelial cancer and the low-grade urinary epithelial cancer by analyzing the patient with the high-grade urinary epithelial cancer and the low-grade urinary epithelial cancer, is favorable for clinically judging the tumor grade, has certain reference value for preoperative evaluation and postoperative follow-up visit, is easy for the distant metastasis and the progress of the muscle layer infiltration type urinary bladder epithelial cancer, for example, can know whether the risk of the invasion of the muscle layer of the bladder wall exists earlier, has great significance for further making clinical diagnosis and treatment strategies, and finds by researching the tumor diameter and the concentration of the ErbB3 protein in the urine, the content of ErbB3 in urine of bladder cancer patients is in moderate positive correlation with the maximum diameter size of tumors, and the concentration of ErbB3 in urine is not correlated with the age of the patients through Spearman correlation analysis, so that the diameter of the tumors can be preliminarily judged through the concentration of ErbB3 in urine.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
- The application of ErbB3 protein in preparing bladder cancer noninvasive diagnosis products is characterized in that: the method comprises the following steps:s1, acquiring medical history and examining physique of a patient suspected to suffer from bladder urothelial cancer;s2, preparing the patient meeting the diagnosis condition before diagnosis;s3, preparing materials required for diagnosis;s4, after the ELISA plate coated with the antibody is balanced to the room temperature, adding 100 mu L of prepared urine samples into corresponding holes;s5, adding a biotin-labeled anti-ErbB 3 antibody into the ELISA plate coated with the antibody;s6, adding diluted HRP-streptavidin into the ELISA plate coated with the antibody;s7, sequentially adding TMB color development solution and stop solution into the ELISA plate coated with the antibody, and reading by using an ELISA reader at 450 nm;s8, calculating a concentration value by adopting sigmaplot12.0 software;s9, comparing the calculated ErbB3 protein concentration value with the ErbB3 protein concentration value of a healthy population, and diagnosing whether the patient has bladder urothelial cancer.
- 2. The use of an ErbB3 protein according to claim 1 in the preparation of a product for the noninvasive diagnosis of urothelial carcinoma of the bladder, wherein: in step S1, the patient who is diagnosed with bladder urothelial cancer needs to satisfy any preliminary diagnosis suspected bladder urothelial cancer, and has not been treated with anti-tumor therapy such as chemotherapy, radiotherapy or surgery; the patient has not suffered from a related disease that may have an effect on urinary protein.
- 3. The use of an ErbB3 protein according to claim 1 in the preparation of a product for the noninvasive diagnosis of urothelial carcinoma of the bladder, wherein: in step S2, after confirming that the patient satisfies all the conditions in S1, morning urine of the next day after patient admission is taken, centrifuged at 4500r/min for 10 minutes at 4 ℃ immediately, and at least 2ml of supernatant is stored in a refrigerator at-80 ℃ for later use.
- 4. The use of an ErbB3 protein according to claim 1 in the preparation of a product for the noninvasive diagnosis of urothelial carcinoma of the bladder, wherein: in step S3, the materials required before diagnosis include: the kit comprises an ELISA plate coated with anti-HumanErbB3 antibody, concentrated 20 Xwashing liquid, deionized water, a plate washing machine, biotin-labeled anti-ErbB 3 antibody, concentrated 200 XHRP-streptavidin, TMB color development liquid, stop solution and a 450nm enzyme labeling instrument; diluting the concentrated washing solution to 20 times by using deionized water for later use; the concentrated HRP-streptavidin was centrifuged and then diluted to 200 fold with deionized water for use.
- 5. The use of an ErbB3 protein according to claim 1 in the preparation of a product for the noninvasive diagnosis of urothelial carcinoma of the bladder, wherein: in the step S4, after the antibody-coated ELISA plate is balanced to room temperature, 100 μ L of the prepared standard and the patient urine sample are added to the corresponding wells, the whole plate strip is sealed by a sealing plate film, and the plate strip is incubated overnight at 4 ℃.
- 6. The use of an ErbB3 protein according to claim 1 in the preparation of a product for the noninvasive diagnosis of urothelial carcinoma of the bladder, wherein: in the step S5, the prepared 1x washing liquid is added to a plate washing machine, the plate washing machine is used for washing the plate strips for 5 times, 300 mu Llx washing liquid is added into each hole, after the plate washing machine is washed clean, 100 mu L of the prepared biotin labeled anti-ErbB 3 antibody is added into each hole of the ELISA plate coated with the antibody, and the incubation is carried out for 1h at room temperature.
- 7. The use of an ErbB3 protein according to claim 1 in the preparation of a product for the noninvasive diagnosis of urothelial carcinoma of the bladder, wherein: in the step S6, the prepared 1x washing liquor is added to a plate washing machine, the plate washing machine is used for washing the lath for 5 times, 300 mu Llx washing liquor is added into each hole, after the plate washing machine is washed clean, 100 mu L of prepared HRP-streptavidin is added into each hole of the ELISA plate coated with the antibody, and the incubation is carried out for 45min at room temperature.
- 8. The use of an ErbB3 protein according to claim 1 in the preparation of a product for the noninvasive diagnosis of urothelial carcinoma of the bladder, wherein: in the step S7, adding the prepared 1x washing liquor to a plate washing machine, washing the plate strips for 5 times by using the plate washing machine, adding 300 mu Llx washing liquor into each hole, after the plate washing machine is cleaned, adding 100 mu LTMB developing solution into each hole of the antibody-coated ELISA plate, incubating for 30min at room temperature in a dark place, adding 50 mu L stop solution into each hole of the antibody-coated ELISA plate, and immediately reading at 450nm of an ELISA reader.
- 9. The use of an ErbB3 protein according to claim 1 in the preparation of a product for the noninvasive diagnosis of urothelial carcinoma of the bladder, wherein: in step S8, the ErbB3 protein concentration value of the urine specimen was calculated using sigmaplot12.0 software.
- 10. The use of an ErbB3 protein according to claim 1 in the preparation of a product for the noninvasive diagnosis of urothelial carcinoma of the bladder, wherein: in step S9, the finally obtained value of the ErbB3 protein concentration in the morning urine of the patient is compared with the value of the ErbB3 protein concentration in the urine of healthy people, and a diagnosis is made as to whether the patient has bladder cancer.
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| CN107223163A (en) * | 2014-12-24 | 2017-09-29 | 豪夫迈·罗氏有限公司 | For the treatment of bladder cancer, diagnosis and method of prognosis |
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