CN106526185B - For detecting the ELISA kit and detection method of castration-resistant prostate cancer - Google Patents
For detecting the ELISA kit and detection method of castration-resistant prostate cancer Download PDFInfo
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- CN106526185B CN106526185B CN201610972678.5A CN201610972678A CN106526185B CN 106526185 B CN106526185 B CN 106526185B CN 201610972678 A CN201610972678 A CN 201610972678A CN 106526185 B CN106526185 B CN 106526185B
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- prostate cancer
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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
- G01N33/57434—Specifically defined cancers of prostate
Abstract
The invention discloses for detecting the ELISA kit and detection method of castration-resistant prostate cancer, including the ELISA ELISA Plates of ACTG1 antibody, β actin antibody and FG gamma antibodies are coated with respectively；Standard items；Sample dilution；ACTG1 antibody, β actin antibody and the FG gamma antibodies of biotin labeling；The horseradish peroxidase of Avidin mark；Cleaning solution；Substrate solution；Reaction terminating liquid and overlay film.Kit of the present invention quick, reasonable simple to operate, can improve the diagnosis efficiency of castration-resistant prostate cancer, have broad application prospects.
Description
Technical field
The present invention relates to immunoassay technology field, more particularly, to detects castration-resistant prostate cancer
ELISA kit and detection method.
Background technology
Prostate cancer is the most common malignant tumour of male urinary system, relatively conventional in American-European countries, is that male is most normal
The malignant tumour seen, account for the second of all malignant tumours.China is the relatively low country of traditional prostate-cancer incidence, still
Recently as the raising of living standards of the people, the change of environment, and the raising of disorder in screening popularity rate, the prostate in China
Cancer morbidity rises year by year.Prostate cancer turns into a kind of important diseases for threatening China's men's health, by more
Come more concern and attention.
One of most important therapeutic modality of prostate cancer is exactly castration therapy, including medicine anti-androgen therapy and operation
Castration, suppress the growth of tumour in a manner of preventing androgen from being attached on androgen receptor.In prostate cancer in early days,
Castration has preferable effect, but after treatment after a while, prostate cancer be gradually converted into androgen it is non-according to
Rely property, no longer sensitive to castration, the state of an illness deteriorates once again, i.e. castration-resistant prostate cancer (CRPC) stage.Castration is supported
The pathogenesis of refractory prostate cancer it is not immediately clear, be prostate cancer basis and clinic also without preferable treatment method
The problem in field.
The diagnosis for castration-resistant prostate cancer does not have good method at present, mainly by monitoring prostate
The change of specific antigen and testosterone, and according to clinical manifestation come comprehensive descision, shortage preferably specificity, it is impossible to exactly
Judge castration-resistant prostate cancer.Early find castration-resistant prostate cancer, the formulation to anaphase strategy and
Prostate cancer is more in depth recognized from molecular level, there is very important meaning.
The content of the invention
The present invention is exactly to solve the product for detecting castration-resistant prostate cancer in the prior art or method specificity
Difference, the problem of accuracy is not high, a kind of ELISA kit and the detection for being used to detect castration-resistant prostate cancer proposed
Method.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is：
For detecting the ELISA kit of castration-resistant prostate cancer, including ACTG1 antibody, β-actin are coated with respectively
The ELISA ELISA Plates of antibody and FG gamma antibodies；Standard items；Sample dilution；ACTG1 antibody, the β-actin of biotin labeling resist
Body and FG gamma antibodies；The horseradish peroxidase of Avidin mark；Cleaning solution；Substrate solution；Reaction terminating liquid and overlay film.
Further, the Sample dilution preparation process is as follows：100mLPBS solution is taken, adds 2g BSA to it, extremely
Final mass concentration is 2%, fully mixes, avoids bubbling.
Further, the preparation process of the cleaning solution is as follows：0.5mL 0.05%Tween-20 are added to 1000mL
In PBS.
Further, it is described to be coated with respectively in the ELISA ELISA Plates of ACTG1 antibody, β-actin antibody and FG gamma antibodies
Coated antibody concentration is 1-100 μ g/mL, the ACTG1 antibody of the biotin labeling, β-actin antibody and FG gamma antibodies it is dense
Spend for 0-100 μ g/mL, the horseradish peroxidase concentration 0.0001-0.0005mg/mL of the Avidin mark, the sample
For the serum of patients with prostate cancer；The mass concentration that the substrate solution is TMB (tetramethyl benzidine) is 0.01%；It is described anti-
It is 2mol/L H to answer terminate liquid2SO4。
Further, it is described to be coated with respectively in the ELISA ELISA Plates of ACTG1 antibody, β-actin antibody and FG gamma antibodies
Coated antibody concentration is preferably 10 μ g/mL.
Further, the concentration of the ACTG1 antibody of the biotin labeling, β-actin antibody and FG gamma antibodies preferably 10 μ
g/mL。
For the detection method for the ELISA kit for detecting castration-resistant prostate cancer, comprise the following steps：
Step 1：Patients with prostate cancer whole blood sample is gathered, 2000rpm 20min centrifugations, supernatant is taken, collects blood
Test tube is disposable apyrogeneity, the test tube of endotoxin-free；
Step 2：Each reagent is balanced to room temperature, testing sample hole and adds the μ L of Sample serum 100, each sample respectively per hole
The hole of Parallel testing 2, while set blank well, each holes of standard control, take standard items β-actin 10ng, ACTG1 2000pg,
Each 100 μ L of FG γ 1000ng are separately added into reacting hole, and blank control wells only add 100 μ L sample dilutions；ELISA Plate, which adds, to be covered
Film, 37 DEG C of incubation 90min；
Step 3：Liquid in hole is discarded, is dried；Three detection plates be separately added into per hole biotin labeling ACTG1 antibody,
β-actin antibody and each 100 μ L of FG gamma antibodies, ELISA Plate add overlay film, 37 DEG C of incubation 1h；
Step 4：Liquid in hole is discarded, is dried, washing ELISA Plate 1-5 times, soaks 1-2min with cleaning solution every time, then get rid of
It is dry；
Step 5：The μ L of horseradish peroxidase 100 of Avidin mark are added per hole, ELISA Plate adds overlay film, 37 DEG C of incubations
30min；
Step 6：Liquid in hole is discarded, is dried, washing ELISA Plate 3-7 times, soaks 1-2min with cleaning solution every time, then get rid of
It is dry；
Step 7：Substrate solution (TMB) 90 μ L are added per hole, ELISA Plate adds overlay film, and 37 DEG C of lucifuges are incubated 15-30min；
Step 8：Per hole add the μ L of reaction terminating liquid 50, terminating reaction, now blueness be changed into yellow；
Step 9：Optical density (the OD in each hole is measured under 450nm wavelength with ELIASA (SPECTRA max plus384)
Value)；
Step 10：Serum sample ACTG1, β-actin, FG γ detected value are calculated according to the OD values of standard items；
The detected value of three indexs is substituted into logistic models：Logit P=0.000014*ACTG1+0.029528*
β-actin+0.000013*FG γ -12.705065, calculate P values, and judge its magnitude relationship with decision threshold.
Further, the step 4 washs ELISA Plate 3 times, soaks 1.5min every time；The step 6 washs ELISA Plate 5
It is secondary, 1.5min is soaked every time.
Further, the decision threshold P is 0.5171589~0.6320831.
Further, the decision threshold P is 0.5746210.
If P values are more than or equal to decision threshold, judge that patient has progressed to castration-resistant prostate cancer, if P values are small
Then judge that patient does not progress to castration-resistant prostate cancer in decision threshold.
The beneficial effects of the present invention are：Effective solve of the invention detects castration-resistant prostate in the prior art
The product or method poor specificity of cancer, the problem of accuracy is not high.Kit of the present invention according to ACTG1, β-actin in serum,
FG γ content diagnoses castration-resistant prostate cancer, has higher sensitivity and a specificity.Kit operation of the present invention
Simple and quick, reasonable, the diagnosis efficiency of castration-resistant prostate cancer can be improved, is had broad application prospects.
Brief description of the drawings
Fig. 1 is that the result of present invention detection serum sample draws figure.
Fig. 2 is the ROC curve of castration-resistant prostate cancer of the present invention diagnosis.
Embodiment
With reference to specific embodiment, the present invention is further detailed explanation.
With reference to embodiment, the present invention is described in further detail, but the invention is not restricted to given implementation
Example：
Embodiment 1：
For detecting the ELISA kit of castration-resistant prostate cancer, including ACTG1 antibody, β-actin are coated with respectively
The ELISA ELISA Plates of antibody and FG gamma antibodies；Standard items；Sample dilution；ACTG1 antibody, the β-actin of biotin labeling resist
Body and FG gamma antibodies；The horseradish peroxidase of Avidin mark；Cleaning solution；Substrate solution；Reaction terminating liquid and overlay film.
Sample dilution preparation process is as follows：100mLPBS solution is taken, 2g BSA is added to it, is to final mass concentration
2%, fully mix, avoid bubbling.
The preparation process of cleaning solution is as follows：0.5mL 0.05%Tween-20 are added in 1000mL PBSs.
The coated antibody concentration being coated with respectively in the ELISA ELISA Plates of ACTG1 antibody, β-actin antibody and FG gamma antibodies
For 10 μ g/mL.
The μ g/mL of concentration 10 of the ACTG1 antibody of biotin labeling, β-actin antibody and FG gamma antibodies.
For the detection method for the ELISA kit for detecting castration-resistant prostate cancer, comprise the following steps：
Step 1：Patients with prostate cancer whole blood sample is gathered, 2000rpm 20min centrifugations, supernatant is taken, collects blood
Test tube is disposable apyrogeneity, the test tube of endotoxin-free；
Step 2：Each reagent is balanced to room temperature, testing sample hole and adds the μ L of Sample serum 100, each sample respectively per hole
The hole of Parallel testing 2, while set blank well, each holes of standard control, take standard items β-actin 10ng, ACTG1 2000pg,
Each 100 μ L of FG γ 1000ng are separately added into reacting hole, and blank control wells only add 100 μ L sample dilutions；ELISA Plate, which adds, to be covered
Film, 37 DEG C of incubation 90min；
Step 3：Liquid in hole is discarded, is dried；Three detection plates be separately added into per hole biotin labeling ACTG1 antibody,
β-actin antibody and each 100 μ L of FG gamma antibodies, ELISA Plate add overlay film, 37 DEG C of incubation 1h；
Step 4：Liquid in hole is discarded, is dried, washing ELISA Plate 3 times, soaks 1.5min with cleaning solution every time, then dry；
Step 5：The μ L of horseradish peroxidase 100 of Avidin mark are added per hole, ELISA Plate adds overlay film, 37 DEG C of incubations
30min；
Step 6：Liquid in hole is discarded, is dried, washing ELISA Plate 5 times, soaks 1.5min with cleaning solution every time, then dry；
Step 7：Substrate solution (TMB) 90 μ L are added per hole, ELISA Plate adds overlay film, and 37 DEG C of lucifuges are incubated 15min, work as mark
When there is obvious gradient in quasi- hole, you can terminate；
Step 8：Per hole add the μ L of reaction terminating liquid 50, terminating reaction, now blueness be changed into yellow；
Step 9：Optical density (the OD in each hole is measured under 450nm wavelength with ELIASA (SPECTRA max plus384)
Value)；
Step 10：Serum sample ACTG1, β-actin, FG γ detected value are calculated according to the OD values of standard items；
The detected value of three indexs is substituted into logistic models：Logit P=0.000014*ACTG1+0.029528*
β-actin+0.000013*FG γ -12.705065, calculate P values, and judge its magnitude relationship with decision threshold.
Decision threshold P is 0.5746210, if P values are more than or equal to 0.5746210, judges that patient has progressed to castration
Repellence prostate cancer, judge that patient does not progress to castration-resistant prostate cancer if P values are less than 0.5746210.In this reality
In 85 samples for applying example, the sensitiveness that castration-resistant prostate cancer is diagnosed using kit of the present invention is 78%, specificity
For 90.9%.
The inspection value of the serum sample of the present embodiment is drawn in Fig. 1, and two groups are compared P ＜ 0.05, and difference has statistics
Meaning.
Fig. 2 is the ROC curve that P values diagnose as castration-resistant prostate cancer, and abscissa is 1- specificities, and ordinate is
Sensitivity, wherein AUC=0.902, ROC represent TG-AUC.It can be seen that sensitivity reaches 78%, specificity
Reach 90.9%.
It can be seen that the content of ACTG1, β-actin, FG γ in serum of patients with prostate cancer are detected using the present invention, can be quick
Castration-resistant prostate cancer is diagnosed exactly, and there is higher sensitivity and specificity.
Claims (6)
1. the ELISA kit for detecting castration-resistant prostate cancer, it is characterised in that resist including being coated with ACTG1 respectively
The ELISA ELISA Plates of body, β-actin antibody and FG gamma antibodies；Standard items；Sample dilution；The ACTG1 of biotin labeling resists
Body, β-actin antibody and FG gamma antibodies；The horseradish peroxidase of Avidin mark；Cleaning solution；Substrate solution；Reaction terminating
Liquid and overlay film.
2. it is used for the ELISA kit for detecting castration-resistant prostate cancer as claimed in claim 1, it is characterised in that described
Sample dilution preparation process is as follows：100mLPBS solution is taken, 2g BSA are added to it, is 2% to final mass concentration, fully
Mix, avoid bubbling.
3. it is used for the ELISA kit for detecting castration-resistant prostate cancer as claimed in claim 1, it is characterised in that described
The preparation process of cleaning solution is as follows：0.5mL 0.05%Tween-20 are added in 1000mL PBSs.
4. it is used for the ELISA kit for detecting castration-resistant prostate cancer as claimed in claim 1, it is characterised in that described
The coated antibody concentration being coated with respectively in the ELISA ELISA Plates of ACTG1 antibody, β-actin antibody and FG gamma antibodies is 1-100 μ
G/mL, the concentration of the ACTG1 antibody of the biotin labeling, β-actin antibody and FG gamma antibodies are≤100 μ g/mL, the parent
With the horseradish peroxidase concentration 0.0001-0.0005mg/mL of element mark, sample is the serum of patients with prostate cancer；It is described
Substrate solution is the TMB (tetramethyl benzidine) that mass concentration is 0.01%；The reaction terminating liquid is 2mol/L H2SO4。
5. it is used for the ELISA kit for detecting castration-resistant prostate cancer as claimed in claim 4, it is characterised in that described
The coated antibody concentration being coated with respectively in the ELISA ELISA Plates of ACTG1 antibody, β-actin antibody and FG gamma antibodies is preferably 10 μ
g/mL。
6. it is used for the ELISA kit for detecting castration-resistant prostate cancer as claimed in claim 4, it is characterised in that described
The concentration preferably 10 μ g/mL of the ACTG1 antibody of biotin labeling, β-actin antibody and FG gamma antibodies.
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Cited By (1)
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| WO2022034343A1 (en) * | 2020-08-14 | 2022-02-17 | Oxford Brookes University | Metastatic biomarker |
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| HUP0303749A3 (en) * | 2001-04-03 | 2005-09-28 | Merck Patent Gmbh | Renal cell carcinoma tumor markers |
| US7309487B2 (en) * | 2004-02-09 | 2007-12-18 | George Inana | Methods and compositions for detecting and treating retinal diseases |
| DE102006056784A1 (en) * | 2006-12-01 | 2008-06-05 | Meyer, Helmut E., Prof.Dr. | Biomarker for the diagnosis of pancreatic cancer |
| US20110030072A1 (en) * | 2008-12-04 | 2011-02-03 | Sigma-Aldrich Co. | Genome editing of immunodeficiency genes in animals |
| BR112012025196A2 (en) * | 2010-04-02 | 2017-03-28 | Veridex Llc | -based prognosis of prostate-specific antigen (psa) recurrence in patients with clinically localized prostate cancer |
| KR20210012064A (en) * | 2012-07-27 | 2021-02-02 | 아라곤 파마슈티컬스, 인코포레이티드 | Methods and compositions for determining resistance to androgen receptor therapy |
| CN105779599B (en) * | 2016-04-05 | 2020-09-11 | 上海美吉生物医药科技有限公司 | Kit for detecting metastatic castration resistant prostate cancer drug resistance |
| CN105861681A (en) * | 2016-05-03 | 2016-08-17 | 中山大学附属第医院 | Molecular index, kit and method for sample quality control before lung cancer EGFR gene Sanger sequencing |
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