CN106405085A - ELISA kit used for detecting castration-resistant prostate cancer and using method thereof - Google Patents
ELISA kit used for detecting castration-resistant prostate cancer and using method thereof Download PDFInfo
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- 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
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- G01N33/57434—Specifically defined cancers of prostate
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Abstract
The invention discloses an ELISA kit used for detecting castration-resistant prostate cancer and a using method thereof, and relates to immunoassays. The kit disclosed by the invention comprises a beta-actin antibody, and is capable of detecting the beta-actin content in serum by virtue of a double-antibody sandwich ELISA method and further judging whether the disease of a patient has developed as the castration-resistant prostate cancer. According to the invention, the problem in the prior art that a product or a method used for detecting the castration-resistant prostate cancer is low in specificity and low in accuracy is effectively solved. The kit disclosed by the invention is capable of diagnosing the castration-resistant prostate cancer according to the beta-actin content in the serum, and has relatively high sensitivity and specificity. The kit disclosed by the invention is simple and rapid in operation, reasonable and feasible, is capable of improving the diagnostic efficiency of the castration-resistant prostate cancer and has wide application prospects.
Description
Technical field
The present invention relates to immunoassay, specifically a kind of ELISA reagent for detecting castration-resistant prostate cancer
Box and using method.
Background technology
Prostate cancer is the modal malignant tumour of male urinary system, relatively conventional in American-European countries, is that the male sex is the most normal
The malignant tumour seen, accounts for the second of all malignant tumours.The relatively low country of prostate-cancer incidence that China is traditional, but
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 of China
Cancer morbidity rises year by year.Prostate cancer has become a kind of important diseases threatening China's men's health, is being subject to more
Come more concerns and attention.
One of most important therapeutic modality of prostate cancer is exactly castration therapy, including medicine anti-androgen therapy and operation
Castration, to suppress the growth of tumour in the way of stoping androgen from being attached on androgen receptor.In prostate cancer early stage,
Castration has preferable effect, but through treatment after a while after, prostate cancer be gradually converted into androgen non-according to
Bad 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, does not also have preferable treatment method, is prostate cancer basis and clinic
The difficult problem in field.
Diagnosis for castration-resistant prostate cancer does not have good method at present, mainly relies on monitoring prostate
Specific antigen and the change of testosterone, and according to clinical manifestation come comprehensive descision, lack preferably specificity it is impossible to exactly
Judge castration-resistant prostate cancer.Early find castration-resistant prostate cancer, to the formulation of anaphase strategy and
Prostate cancer is more in depth recognized from molecular level, has very important meaning.
Content of the invention
The present invention is exactly to solve to detect the product of castration-resistant prostate cancer or method specificity in prior art
Difference, accuracy not high problem, a kind of ELISA kit for detecting castration-resistant prostate cancer being proposed and use
Method.
The present invention is to realize according to technical scheme below.
A kind of ELISA kit for detecting castration-resistant prostate cancer, described kit includes β-actin and resists
Detection reagent needed for body, and double antibody sandwich ELISA.
A kind of ELISA kit for detecting castration-resistant prostate cancer, described kit includes being coated β-actin
The ELISA ELISA Plate of antibody, standard items, standard items ＆ sample diluting liquid, the β-actin antibody of biotin labeling, Avidin mark
One or more of the horseradish peroxidase of note, cleaning solution, substrate solution, reaction terminating liquid, overlay film.
Detection sample is the serum of patients with prostate cancer.
A kind of using method of the above-mentioned ELISA kit for detecting castration-resistant prostate cancer, walks including following
Suddenly：
I. collection patients with prostate cancer whole blood sample, centrifuging and taking supernatant；
II. set blank well, gauge orifice, testing sample hole respectively, blank well adds standard items sample diluting liquid, remaining hole mark-on respectively
Quasi- product or testing sample, mix；ELISA Plate adds overlay film, incubation；
III. discard in the hole liquid, dry；Every hole adds the β-actin antibody of biotin labeling, and ELISA Plate adds overlay film, incubates；
IV. discard in the hole liquid, dry, wash ELISA Plate, then dry；
V. every hole adds the horseradish peroxidase of Avidin mark, and ELISA Plate adds overlay film, incubates；
VI. discard in the hole liquid, dry, wash ELISA Plate, then dry；
VII. every hole adds substrate solution, and ELISA Plate adds overlay film, and lucifuge is incubated；
VIII. every hole adds reaction terminating liquid；
Ⅸ. measure the OD value in each hole under 450nm wavelength；
Ⅹ. the OD value according to standard items calculates the detected value of serum sample β-actin, and judges that it is closed with the size of decision threshold
System.
In described step I whole blood sample in room temperature place 0 ~ 4 hour or 0 ~ 8 DEG C overnight after 500 ~ 1500 × g be centrifuged 10 ~
30 minutes.
Washing ELISA Plate 1 ~ 5 time in described step IV, soaks 1-2 minute every time；Washing ELISA Plate 3 ~ 7 times in step VI,
Soak 1-2 minute every time.
In described step IV, washing ELISA Plate 3 times, soak 1.5 minutes every time；Washing ELISA Plate 5 times in step VI, every time
Soak 1.5 minutes.
Described decision threshold is 350.98mg/ml ~ 438.88 mg/ml.
Described decision threshold is 389.98mg/ml.
Present invention obtains following beneficial effect.
The present invention effectively solves the product detecting castration-resistant prostate cancer in prior art or method specificity
The not high problem of difference, accuracy.Kit of the present invention diagnoses castration-resistant prostate according to the content of β-actin in serum
Cancer, has higher sensitivity and specificity.Kit of the present invention quick, reasonable simple to operate, can improve castration opposing
The diagnosis efficiency of property prostate cancer, has broad application prospects.
Brief description
Fig. 1 is the non-castration-resistant prostate cancer of the present invention and castration-resistant prostate cancer serum beta-actin
The statistical result figure of detected value；
Fig. 2 is that the present invention to diagnose the ROC curve figure of castration-resistant prostate cancer with serumβ-actin level.
Specific embodiment
Below in conjunction with the accompanying drawings and embodiment the present invention is described further.
A kind of ELISA kit for detecting castration-resistant prostate cancer, described kit includes β-actin and resists
Detection reagent needed for body, and double antibody sandwich ELISA.
A kind of ELISA kit for detecting castration-resistant prostate cancer, described kit includes being coated β-actin
The ELISA ELISA Plate of antibody, standard items, standard items ＆ sample diluting liquid, the β-actin antibody of biotin labeling, Avidin mark
One or more of the horseradish peroxidase of note, cleaning solution, substrate solution, reaction terminating liquid, overlay film.
Detection sample is the serum of patients with prostate cancer.
A kind of using method of the above-mentioned ELISA kit for detecting castration-resistant prostate cancer, walks including following
Suddenly：
I. collection serum of patients with prostate cancer：Whole blood sample is placed in room temperature and is overnight centrifuged 20 after 1000 × g in 2 hours or 4 DEG C
Minute, take supernatant can detect, the test tube of collect blood should be disposable apyrogeneity, endotoxin-free test tube.
Before experiment starts, each reagent all should balance to room temperature；When reagent or sample preparation, it is both needed to fully mix, and as far as possible
Avoid bubbling.
It is coated the ELISA ELISA Plate of β-actin antibody：The concentration of coated antibody is 1～100 μ g/ml, preferably 10 μ g/
ml.
Standard items：β-actin 10 ng, is configured to following concentration： 10、5、2.5、1.25、0.625、0.313、0.156、
0ng/mL.
Standard items ＆ sample diluting liquid：100ml PBS, adds BSA 2g, to final concentration 2%
β-actin the antibody of biotin labeling：0～100 μ g/ml, preferably 10 μ g/ml.
The horseradish peroxidase of Avidin mark：0.0001～0.0005 mg/ml
Cleaning solution:0.05%Tween-20 0.5ml adds in PBS 1000ml.
Substrate solution：TMB (tetramethyl benzidine).
Reaction terminating liquid：2M H2SO4.
II. sample-adding：Set blank well, gauge orifice, testing sample hole respectively.Blank well adds standard items sample diluting liquid 100 μ
L, remaining hole adds standard items or testing sample 100 μ L respectively, has been careful not to bubble, during sample-adding, sample has been added on ELISA Plate bottom,
Do not touch hole wall as far as possible, gently rock mixing.To ELISA Plate overlay film, 37 DEG C are incubated 90 minutes.
III. discard liquid, dry, without washing.β-actin antibody 100 μ the L of biotin labeling is added in each hole,
ELISA Plate adds overlay film, and 37 DEG C incubate 1 hour.
IV. discard in the hole liquid, dry, wash plate 3 times, soak 1-2 minute, the every hole of about 350 μ L/ every time, dry
And pat on blotting paper in the hole liquid is patted dry.
V. every hole adds the horseradish peroxidase of Avidin mark, adds overlay film, and 37 DEG C incubate 30 minutes.
VI. discard in the hole liquid, dry, wash plate 5 times, method is with step IV.
VII. every hole adds substrate solution (TMB) 90 μ L, and ELISA Plate adds that 37 DEG C of lucifuges of overlay film are incubated 15 minutes about
（Take the circumstances into consideration to shorten according to actual colour developing situation or extend, but may not exceed 30 minutes.When obvious gradient in gauge orifice, that is,
Can terminate）.
VIII. every hole adds reaction terminating liquid 50 μ L, terminating reaction, and now blueness is vertical turns yellow.The addition sequence of terminate liquid
Should try one's best identical with the addition sequence of substrate solution.
Ⅸ. use ELIASA immediately（SPECTRA max plus384）Optical density in each hole of 450nm wavelength measurement（OD
Value）.
Ⅹ. the OD value according to standard items substitutes into the detected value that equation calculates serum sample β-actin, and judges it and sentence
Determine the magnitude relationship of threshold value.
If β-actin detected value is more than or equal to decision threshold, judge that patient has progressed to castration-resistant prostate
Cancer, if β-actin detected value is less than decision threshold, judges that patient does not progress to castration-resistant prostate cancer.With β in serum-
Actin detected value is 350.98mg/ml ~ 438.88 mg/ml, preferred 389.98mg/ml is decision threshold.
Embodiment 1
The present embodiment is with β-actin detected value 389.98mg/ml as judgment threshold, if β-actin detected value is more than or equal to
During 389.98mg/ml, then judge that patient progresses to castration-resistant prostate cancer, if β-actin detected value is less than
389.98mg/ml then judges that patient does not progress to castration-resistant prostate cancer.In 87 samples of the present embodiment, using this
The sensitiveness that invention kit diagnoses castration-resistant prostate cancer is 72.1%, and specificity is 93.2%.
The inspection value of the serum sample of the present embodiment is drawn in FIG, and two groups are compared P<0.05, difference has statistics meaning
Justice.
The ROC curve that Fig. 2 diagnoses as castration-resistant prostate cancer for β-actin, abscissa is 1- specificity, indulges and sits
It is designated as sensitivity, AUC=0.884, wherein ROC represent TG-AUC.It can be seen that sensitivity reaches 72.1%, spy
Different degree reaches 93.2%.
It can be seen that, the content that β-actin in serum of patients with prostate cancer is detected using the present invention, can rapidly and accurately diagnose
Gesture repellence prostate cancer, has higher sensitivity and specificity.
Claims (9)
1. a kind of ELISA kit for detecting castration-resistant prostate cancer it is characterised in that：Described kit include β-
Actin antibody, and the detection reagent needed for double antibody sandwich ELISA.
2. a kind of ELISA kit for detecting castration-resistant prostate cancer according to claim 1, its feature exists
In：Described kit includes being coated the ELISA ELISA Plate of β-actin antibody, standard items, standard items ＆ sample diluting liquid, biology
β-actin the antibody of element mark, the horseradish peroxidase of Avidin mark, cleaning solution, substrate solution, reaction terminating liquid, cover
One or more of film.
3. a kind of ELISA kit for detecting castration-resistant prostate cancer according to claim 1, its feature exists
In：Detection sample is the serum of patients with prostate cancer.
4. it is used for described in a kind of claim 1-3 detecting the using method of the ELISA kit of castration-resistant prostate cancer, its
It is characterised by：Comprise the following steps：
I. collection patients with prostate cancer whole blood sample, centrifuging and taking supernatant；
II. set blank well, gauge orifice, testing sample hole respectively, blank well adds standard items sample diluting liquid, remaining hole mark-on respectively
Quasi- product or testing sample, mix；ELISA Plate adds overlay film, incubation；
III. discard in the hole liquid, dry；Every hole adds the β-actin antibody of biotin labeling, and ELISA Plate adds overlay film, incubates；
IV. discard in the hole liquid, dry, wash ELISA Plate, then dry；
V. every hole adds the horseradish peroxidase of Avidin mark, and ELISA Plate adds overlay film, incubates；
VI. discard in the hole liquid, dry, wash ELISA Plate, then dry；
VII. every hole adds substrate solution, and ELISA Plate adds overlay film, and lucifuge is incubated；
VIII. every hole adds reaction terminating liquid；
Ⅸ. measure the OD value in each hole under 450nm wavelength；
Ⅹ. the OD value according to standard items calculates the detected value of serum sample β-actin, and judges that it is closed with the size of decision threshold
System.
5. the user of a kind of ELISA kit for detecting castration-resistant prostate cancer according to claim 4
Method it is characterised in that：In described step I, whole blood sample places 0 ~ 4 hour or 0 ~ 8 DEG C overnight after 500 ~ 1500 × g in room temperature
Centrifugation 10 ~ 30 minutes.
6. the user of a kind of ELISA kit for detecting castration-resistant prostate cancer according to claim 4
Method it is characterised in that：Washing ELISA Plate 1 ~ 5 time in described step IV, soaks 1-2 minute every time；ELISA Plate 3 is washed in step VI
~ 7 times, soak 1-2 minute every time.
7. the user of a kind of ELISA kit for detecting castration-resistant prostate cancer according to claim 6
Method it is characterised in that：In described step IV, washing ELISA Plate 3 times, soak 1.5 minutes every time；ELISA Plate 5 is washed in step VI
Secondary, soak 1.5 minutes every time.
8. the user of a kind of ELISA kit for detecting castration-resistant prostate cancer according to claim 4
Method it is characterised in that：Described decision threshold is 350.98mg/ml ~ 438.88 mg/ml.
9. the user of a kind of ELISA kit for detecting castration-resistant prostate cancer according to claim 8
Method it is characterised in that：Described decision threshold is 389.98mg/ml.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610770859.XA CN106405085B (en) | 2016-08-31 | 2016-08-31 | For detecting the ELISA kit and application method of castration-resistant prostate cancer |
Applications Claiming Priority (1)
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|---|---|---|---|
| CN201610770859.XA CN106405085B (en) | 2016-08-31 | 2016-08-31 | For detecting the ELISA kit and application method of castration-resistant prostate cancer |
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| Publication Number | Publication Date |
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| CN106405085A true CN106405085A (en) | 2017-02-15 |
| CN106405085B CN106405085B (en) | 2018-03-27 |
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| WO2011127219A1 (en) * | 2010-04-06 | 2011-10-13 | Caris Life Sciences Luxembourg Holdings | Circulating biomarkers for disease |
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2016
- 2016-08-31 CN CN201610770859.XA patent/CN106405085B/en active Active
Patent Citations (6)
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|---|---|---|---|---|
| WO2011127219A1 (en) * | 2010-04-06 | 2011-10-13 | Caris Life Sciences Luxembourg Holdings | Circulating biomarkers for disease |
| CN102690787A (en) * | 2012-04-13 | 2012-09-26 | 湖南师范大学 | Hybridoma cells capable of secreting an anti human [beta]-actin protein monoclonal antibody and preparation method thereof |
| CN104685072A (en) * | 2012-07-27 | 2015-06-03 | 阿拉贡药品公司 | Methods and compositions for determining resistance to androgen receptor therapy |
| WO2014206259A1 (en) * | 2013-06-24 | 2014-12-31 | 复旦大学附属肿瘤医院 | Use of prostate cancer gene marker in marking recurrence and metastasis of prostate cancer and method thereof |
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