CN106405085B - For detecting the ELISA kit and application method of castration-resistant prostate cancer - Google Patents
For detecting the ELISA kit and application method of castration-resistant prostate cancer Download PDFInfo
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- CN106405085B CN106405085B CN201610770859.XA CN201610770859A CN106405085B CN 106405085 B CN106405085 B CN 106405085B CN 201610770859 A CN201610770859 A CN 201610770859A CN 106405085 B CN106405085 B CN 106405085B
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- 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
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Abstract
The invention discloses a kind of ELISA kit and application method for being used to detect castration-resistant prostate cancer, it is related to immunoassay.Kit of the present invention includes β actin antibody, and detects the β actin contents in serum by double antibody sandwich ELISA, determines whether patient has progressed to castration-resistant prostate cancer.The effective product for solving the problems, such as to detect castration-resistant prostate cancer in the prior art of the invention or method poor specificity, accuracy be not high.Kit of the present invention diagnoses castration-resistant prostate cancer according to the content of β actin in serum, has higher sensitivity and specificity.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, specifically a kind of ELISA reagents for being used to detect castration-resistant prostate cancer
Box and application 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 use for being used to detect castration-resistant prostate cancer proposed
Method.
The present invention is realized according to following technical scheme.
A kind of ELISA kit for being used to detect castration-resistant prostate cancer, the kit resist including β-actin
Body, and the detection reagent needed for double antibody sandwich ELISA.
A kind of ELISA kit for being used to detect castration-resistant prostate cancer, the kit include coating β-actin
ELISA ELISA Plates, standard items, standard items ＆ sample diluting liquids, β-actin antibody, the Avidin mark of biotin labeling of antibody
One or more in the horseradish peroxidase of note, cleaning solution, substrate solution, reaction terminating liquid, overlay film.
Detect the serum that sample is patients with prostate cancer.
A kind of above-mentioned application method for being used to detect the ELISA kit of castration-resistant prostate cancer, including following step
Suddenly：
I, gathers patients with prostate cancer whole blood sample, centrifuging and taking supernatant；
II, sets blank well, gauge orifice, testing sample hole respectively, and blank well adds standard items sample diluting liquid, remaining hole difference
Add standard items or testing sample, mix；ELISA Plate adds overlay film, is incubated；
III, discards liquid in hole, dries；β-actin the antibody of biotin labeling is added per hole, ELISA Plate adds overlay film, temperature
Educate；
IV, discards liquid in hole, dries, and washs ELISA Plate, then dry；
V, adds the horseradish peroxidase of Avidin mark per hole, and ELISA Plate adds overlay film, incubates；
VI, discards liquid in hole, dries, and washs ELISA Plate, then dry；
VII, adds substrate solution per hole, and ELISA Plate adds overlay film, and lucifuge is incubated；
VIII, adds reaction terminating liquid per hole；
The OD values in each hole are measured under Ⅸ, 450nm wavelength；
Ⅹ, calculates serum sample β-actin detected value according to the OD values of standard items, and judges that it is big with decision threshold
Small relation.
In the step I whole blood sample in room temperature place 0 ~ 4 hour or 0 ~ 8 DEG C overnight after 500 ~ 1500 × g centrifuge 10 ~
30 minutes.
ELISA Plate is washed in the step IV 1 ~ 5 time, soak 1-2 minutes every time；ELISA Plate is washed in step VI 3 ~ 7 times,
Immersion 1-2 minutes every time.
ELISA Plate is washed in the step IV 3 times, every time immersion 1.5 minutes；ELISA Plate is washed in step VI 5 times, every time
Immersion 1.5 minutes.
The decision threshold is the mg/ml of 350.98mg/ml ~ 438.88.
The decision threshold is 389.98mg/ml.
Present invention obtains following beneficial effect.
It is of the invention effectively 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.Kit of the present invention diagnoses castration-resistant prostate according to the content of β-actin in serum
Cancer, there is higher sensitivity and specificity.Kit of the present invention quick, reasonable simple to operate, can improve castration resistance
The diagnosis efficiency of property prostate cancer, has broad application prospects.
Brief description of the drawings
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 the ROC curve figure that the present invention diagnoses castration-resistant prostate cancer with serumβ-actin levels.
Embodiment
Below in conjunction with the accompanying drawings and embodiment the present invention is described further.
A kind of ELISA kit for being used to detect castration-resistant prostate cancer, the kit resist including β-actin
Body, and the detection reagent needed for double antibody sandwich ELISA.
A kind of ELISA kit for being used to detect castration-resistant prostate cancer, the kit include coating β-actin
ELISA ELISA Plates, standard items, standard items ＆ sample diluting liquids, β-actin antibody, the Avidin mark of biotin labeling of antibody
One or more in the horseradish peroxidase of note, cleaning solution, substrate solution, reaction terminating liquid, overlay film.
Detect the serum that sample is patients with prostate cancer.
A kind of above-mentioned application method for being used to detect the ELISA kit of castration-resistant prostate cancer, including following step
Suddenly：
I, gathers serum of patients with prostate cancer：Whole blood sample in room temperature place 2 hours or 4 DEG C overnight after 1000 × g from
The heart 20 minutes, take supernatant i.e. detectable, the test tube for collecting blood should be disposable apyrogeneity, endotoxin-free test tube.
Before experiment starts, each reagent all should be balanced 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 with the ELISA ELISA Plates of β-actin antibody：The concentration of coated antibody is 1~100 μ g/ml, preferably 10 μ g/
ml。
Standard items：The ng of β-actin 10, are configured to following concentration： 10、5、2.5、1.25、0.625、0.313、0.156、
0ng/mL。
Standard items ＆ sample diluting liquids：100ml PBS, BSA 2g are added, 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 are added in PBS 1000ml.
Substrate solution：TMB (tetramethyl benzidine).
Reaction terminating liquid：2M H2SO4。
II, is loaded：Blank well, gauge orifice, testing sample hole are set respectively.Blank well adds the μ of standard items sample diluting liquid 100
L, remaining hole add standard items or the μ L of testing sample 100 respectively, have been careful not to bubble, and sample is added on into ELISA Plate bottom during sample-adding,
Hole wall is not touched as far as possible, gently rocks mixing.ELISA Plate overlay film is given, 37 DEG C are incubated 90 minutes.
III, discards liquid, dries, without washing.The μ L of β-actin antibody 100 of biotin labeling are added in each hole,
ELISA Plate adds overlay film, and 37 DEG C incubate 1 hour.
IV, discards liquid in hole, dries, board-washing 3 times, soaks every time 1-2 minutes, and about 350 μ L/ dry per hole
And patted on blotting paper and pat dry liquid in hole.
V, adds the horseradish peroxidase that Avidin marks per hole, and plus overlay film, 37 DEG C incubate 30 minutes.
VI, discards liquid in hole, dries, board-washing 5 times, method is the same as step IV.
VII, adds the μ L of substrate solution (TMB) 90 per hole, and ELISA Plate is incubated 15 minutes or so plus 37 DEG C of lucifuges of overlay film
（Take the circumstances into consideration to shorten or extend according to actual colour developing situation, but may not exceed 30 minutes.When obvious gradient occurs in gauge orifice, i.e.,
It can terminate）.
VIII, adds the μ L of reaction terminating liquid 50 per hole, and terminating reaction, now blueness is vertical turns yellow.The addition sequence of terminate liquid
It should try one's best identical with the addition sequence of substrate solution.
Ⅸ, uses ELIASA immediately（SPECTRA max plus384）Optical density in each hole of 450nm wavelength measurements（OD
Value）.
Ⅹ, substitutes into equation according to the OD values of standard items and calculates serum sample β-actin detected value, and judges it with sentencing
Determine the magnitude relationship of threshold value.
If β-actin detected values are more than or equal to decision threshold, judge that patient has progressed to castration-resistant prostate
Cancer, judge that patient does not progress to castration-resistant prostate cancer if β-actin detected values are less than decision threshold.With β in serum-
Actin detected values are that the mg/ml, preferably 389.98mg/ml of 350.98mg/ml ~ 438.88 is decision threshold.
Embodiment 1
The present embodiment is using β-actin detected values 389.98mg/ml as judgment threshold, if β-actin detected values are more than or equal to
During 389.98mg/ml, then judge that patient has progressed to castration-resistant prostate cancer, if β-actin detected values are less than
389.98mg/ml then judges that patient does not progress to castration-resistant prostate cancer.In 87 samples of the present embodiment, this is used
The sensitiveness of invention kit diagnosis 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. 1, and two groups are compared P<0.05, difference has statistics meaning
Justice.
Fig. 2 is the ROC curve that β-actin diagnose as castration-resistant prostate cancer, and abscissa is 1- specificities, indulges and sits
Sensitivity is designated as, 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 detecting the content of β-actin in serum of patients with prostate cancer using the present invention, can rapidly and accurately diagnose
Gesture repellence prostate cancer, there is higher sensitivity and specificity.
Claims (7)
1. β-actin antibody is preparing the purposes in being used to detect the ELISA kit of castration-resistant prostate cancer, its feature
It is：The kit includes β-actin antibody, and the content of β-actin in serum is detected using double-antibodies sandwich ELISA
To detect castration-resistant prostate cancer, decision threshold is the mg/ml of 350.98mg/ml ~ 438.88.
2. β-actin antibody according to claim 1 is preparing the ELISA examinations for detecting castration-resistant prostate cancer
Purposes in agent box, it is characterised in that：The kit includes ELISA ELISA Plates, standard items, the mark of coating β-actin antibody
Quasi- product ＆ sample diluting liquids, the β-actin antibody of biotin labeling, the horseradish peroxidase of Avidin mark, cleaning solution, bottom
One or more in thing solution, reaction terminating liquid, overlay film.
3. β-actin antibody according to claim 1 is preparing the ELISA examinations for detecting castration-resistant prostate cancer
Purposes in agent box, it is characterised in that：The user for being used to detect the ELISA kit of castration-resistant prostate cancer
Method, comprise the following steps：
I, gathers patients with prostate cancer whole blood sample, centrifuging and taking supernatant；
II, sets blank well, gauge orifice, testing sample hole respectively, and blank well adds standard items sample diluting liquid, and mark-on is distinguished in remaining hole
Quasi- product or testing sample, mix；ELISA Plate adds overlay film, is incubated；
III, discards liquid in hole, dries；β-actin the antibody of biotin labeling is added per hole, ELISA Plate adds overlay film, incubates；
IV, discards liquid in hole, dries, and washs ELISA Plate, then dry；
V, adds the horseradish peroxidase of Avidin mark per hole, and ELISA Plate adds overlay film, incubates；
VI, discards liquid in hole, dries, and washs ELISA Plate, then dry；
VII, adds substrate solution per hole, and ELISA Plate adds overlay film, and lucifuge is incubated；
VIII, adds reaction terminating liquid per hole；
The OD values in each hole are measured under Ⅸ, 450nm wavelength；
Ⅹ, calculates serum sample β-actin detected value according to the OD values of standard items, and judges that the size of itself and decision threshold is closed
System, the decision threshold are the mg/ml of 350.98mg/ml ~ 438.88.
4. β-actin antibody according to claim 3 is preparing the ELISA examinations for detecting castration-resistant prostate cancer
Purposes in agent box, it is characterised in that：In the step I whole blood sample in room temperature place 0 ~ 4 hour or 0 ~ 8 DEG C overnight after
500 ~ 1500 × g is centrifuged 10 ~ 30 minutes.
5. β-actin antibody according to claim 3 is preparing the ELISA examinations for detecting castration-resistant prostate cancer
Purposes in agent box, it is characterised in that：ELISA Plate is washed in the step IV 1 ~ 5 time, soak 1-2 minutes every time；In step VI
Wash ELISA Plate 3 ~ 7 times, soak 1-2 minutes every time.
6. β-actin antibody according to claim 5 is preparing the ELISA examinations for detecting castration-resistant prostate cancer
Purposes in agent box, it is characterised in that：ELISA Plate is washed in the step IV 3 times, every time immersion 1.5 minutes；Washed in step VI
Wash ELISA Plate 5 times, every time immersion 1.5 minutes.
7. β-actin antibody according to claim 3 is preparing the ELISA examinations for detecting castration-resistant prostate cancer
Purposes in agent box, it is characterised in that：The decision threshold is 389.98mg/ml.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011127219A1 (en) * | 2010-04-06 | 2011-10-13 | Caris Life Sciences Luxembourg Holdings | Circulating biomarkers for disease |
| 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 |
| CN105779599A (en) * | 2016-04-05 | 2016-07-20 | 上海美吉生物医药科技有限公司 | Kit for detecting metastatic castration resistant prostate cancer (mCRPC) drug resistance |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690787B (en) * | 2012-04-13 | 2013-03-13 | 湖南师范大学 | Hybridoma cells capable of secreting an anti human [beta]-actin protein monoclonal antibody and preparation method thereof |
| CN103602720A (en) * | 2013-06-24 | 2014-02-26 | 复旦大学附属肿瘤医院 | Application and method of prostatic cancer genetic marker in marking relapse and metastasis of prostatic cancer |
| CN104237535B (en) * | 2014-09-30 | 2016-02-10 | 天津市泌尿外科研究所 | The application of neurotensin in diagnosis castration-resistant prostate cancer neuroendocrine differentiated hypotype and judging prognosis |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011127219A1 (en) * | 2010-04-06 | 2011-10-13 | Caris Life Sciences Luxembourg Holdings | Circulating biomarkers for disease |
| 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 |
| CN105779599A (en) * | 2016-04-05 | 2016-07-20 | 上海美吉生物医药科技有限公司 | Kit for detecting metastatic castration resistant prostate cancer (mCRPC) drug resistance |
Non-Patent Citations (1)
| Title |
|---|
| 去势抵抗型前列腺癌的研究进展;郑路等;《癌症进展》;20150731;第13卷(第4期);364-368,375 * |
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