CN106198985A - For detecting ELISA kit and the using method of castration-resistant prostate cancer - Google Patents
For detecting ELISA kit and the using method of castration-resistant prostate cancer Download PDFInfo
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- CN106198985A CN106198985A CN201610770860.2A CN201610770860A CN106198985A CN 106198985 A CN106198985 A CN 106198985A CN 201610770860 A CN201610770860 A CN 201610770860A CN 106198985 A CN106198985 A CN 106198985A
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- Prior art keywords
- prostate cancer
- castration
- resistant prostate
- elisa
- hole
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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 a kind of ELISA kit for detecting castration-resistant prostate cancer and using method, relate to immunoassay.Test kit of the present invention includes FG gamma antibodies, and by the FG γ content in double antibody sandwich ELISA detection serum, determines whether that patient has progressed to castration-resistant prostate cancer the most.The problem that the present invention effectively solves in prior art and detects the product of castration-resistant prostate cancer or method poor specificity, accuracy is the highest.Test kit of the present invention diagnoses castration-resistant prostate cancer according to the content of FG γ in serum, has higher sensitivity and specificity.Test kit of the present invention is simple to operate quickly, reasonable, can improve the diagnosis efficiency of castration-resistant prostate cancer, have broad application prospects.
Description
Technical field
The present invention relates to immunoassay, a kind of ELISA reagent for detecting castration-resistant prostate cancer
Box and using method.
Background technology
Carcinoma of prostate is the modal malignant tumor of male urinary system, relatively conventional in American-European countries, is that male is the most normal
The malignant tumor seen, accounts for the second of all malignant tumor.The country that prostate-cancer incidence that China is traditional is relatively low, 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 the most year by year.Carcinoma of prostate has become a kind of important diseases threatening China's men's health, by more
Come the most concerns and attention.
One of most important therapeutic modality of carcinoma of prostate is exactly castration therapy, including medicine anti-androgen therapy and operation
Castration, suppresses the growth of tumor in the way of stoping androgen to be attached on androgen receptor.In carcinoma of prostate in early days,
Castration has preferable effect, but after treatment after a while, carcinoma of prostate is gradually converted into that androgen is non-to be depended on
Relying property, the most 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 be not immediately clear, does not also have preferable Therapeutic Method, is that carcinoma of prostate basis is with clinical
The difficult problem in field.
Diagnosis for castration-resistant prostate cancer at present does not has good method, mainly relies on monitoring prostate
Specific antigen and the change of testosterone, and carry out comprehensive descision according to clinical manifestation, lack preferable specificity, it is impossible to exactly
Judge castration-resistant prostate cancer.Early find castration-resistant prostate cancer, to the formulation of anaphase strategy and
Carcinoma of prostate is more in depth recognized from molecular level, has very important meaning.
Summary of the invention
The present invention is contemplated to product or the method specificity solving to detect castration-resistant prostate cancer in prior art
Difference, problem that accuracy is the highest, a kind of ELISA kit for detecting castration-resistant prostate cancer proposed and use
Method.
The present invention realizes according to techniques below scheme.
A kind of ELISA kit for detecting castration-resistant prostate cancer, described test kit includes FG gamma antibodies, with
And the detectable needed for double antibody sandwich ELISA.
A kind of ELISA kit for detecting castration-resistant prostate cancer, described test kit includes that being coated FG γ resists
The ELISA ELISA Plate of body, standard substance, standard substance & sample diluting liquid, biotin labeled FG gamma antibodies, Avidin labelling peppery
One or more in root peroxidase, cleaning mixture, substrate solution, reaction terminating liquid, overlay film.
Detection sample is the serum of patients with prostate cancer.
The using method of a kind of above-mentioned ELISA kit for detecting castration-resistant prostate cancer, including following step
Rapid:
I. gather patients with prostate cancer whole blood sample, centrifuging and taking supernatant；
II. setting blank well, gauge orifice, testing sample hole respectively, blank well adds standard substance sample diluting liquid, remaining hole mark-on respectively
Quasi-product or testing sample, mixing；ELISA Plate adds overlay film, hatches；
III. discard liquid in hole, dry；Every hole adds biotin labeled FG gamma antibodies, and ELISA Plate adds overlay film, incubation；
IV. discard liquid in hole, dry, detersive enzyme target, then dry；
V. every hole adds the horseradish peroxidase of Avidin labelling, and ELISA Plate adds overlay film, incubation；
VI. discard liquid in hole, dry, detersive enzyme target, then dry；
VII. every hole adds substrate solution, and ELISA Plate adds overlay film, and lucifuge is hatched；
VIII. every hole adds reaction terminating liquid；
Ⅸ. measure the OD value in each hole under 450nm wavelength；
Ⅹ. calculate the detected value of serum sample FG γ according to the OD value of standard substance, and judge the magnitude relationship of itself and decision threshold.
In described step I whole blood sample in room temperature place 0 ~ 4 hour or 0 ~ 8 DEG C overnight after in 500 ~ 1500 × g be centrifuged 10 ~
30 minutes.
Detersive enzyme target 1 ~ 5 time in described step IV, soaks 1-2 minute every time；Detersive enzyme target 3 ~ 7 times in step VI,
Soak 1-2 minute every time.
Detersive enzyme target 3 times in described step IV, soak 1.5 minutes every time；Detersive enzyme target 5 times in step VI, every time
Soak 1.5 minutes.
Described decision threshold is 19748.73mg/ml ~ 24137.33mg/ml.
Described decision threshold is 21943.03mg/ml.
Present invention obtains following beneficial effect.
The present invention effectively solves in prior art product or the method specificity detecting castration-resistant prostate cancer
Difference, the problem that accuracy is the highest.Test kit of the present invention diagnoses castration-resistant prostate cancer according to the content of FG γ in serum,
There is higher sensitivity and specificity.Test kit of the present invention is simple to operate quickly, reasonable, before can improving castration-resistant
The diagnosis efficiency of row adenocarcinoma, has broad application prospects.
Accompanying drawing explanation
Fig. 1 is FG γ detection in the non-castration-resistant prostate cancer of the present invention and castration-resistant prostate cancer patients serum
The statistical result figure of value；
Fig. 2 be the present invention with serum FG γ level to diagnose the ROC curve figure of castration-resistant prostate cancer.
Detailed description of the invention
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 test kit includes FG gamma antibodies, with
And the detectable needed for double antibody sandwich ELISA.
A kind of ELISA kit for detecting castration-resistant prostate cancer, described test kit includes that being coated FG γ resists
The ELISA ELISA Plate of body, standard substance, standard substance & sample diluting liquid, biotin labeled FG gamma antibodies, Avidin labelling peppery
One or more in root peroxidase, cleaning mixture, substrate solution, reaction terminating liquid, overlay film.
Detection sample is the serum of patients with prostate cancer.
The using method of a kind of above-mentioned ELISA kit for detecting castration-resistant prostate cancer, including following step
Rapid:
I. gather serum of patients with prostate cancer: whole blood sample is centrifuged 20 in 1000 × g after room temperature placement 2 hours or 4 DEG C overnight
Minute, taking supernatant can detect, and the test tube collecting blood should be disposable apyrogeneity, without endotoxin 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 FG gamma antibodies: the concentration of coated antibody is 1～100 μ g/ml, preferably 10 μ g/ml.
Standard substance: FG γ 1000ng, are configured to following concentration: 1000,500,250,125,62.5,31.25,
15.625、0ng/mL。
Standard substance & sample diluting liquid: 100ml PBS, adds BSA 2g, to final concentration 2%
Biotin labeled FG gamma antibodies: 0～100 μ g/ml, preferably 10 μ g/ml.
The horseradish peroxidase of Avidin labelling: 0.0001～0.0005 mg/ml
Cleaning mixture: 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 substance sample diluting liquid 100 μ
L, remaining hole adds standard substance or testing sample 100 μ L respectively, has been careful not to bubble, has been added on bottom ELISA Plate by sample during sample-adding,
Do not touch hole wall as far as possible, rock mixing gently.To ELISA Plate overlay film, hatch 90 minutes for 37 DEG C.
III. discard liquid, dry, need not wash.Each hole adds biotin labeled FG gamma antibodies 100 μ L, enzyme
Target adds overlay film, 37 DEG C of incubations 1 hour.
IV. discard liquid in hole, dry, wash plate 3 times, soak 1-2 minute, the about 350 every holes of μ L/ every time, dry
And pat in absorbent paper liquid in hole is patted dry.
V. every hole adds the horseradish peroxidase 100 μ L of Avidin labelling, adds overlay film, 37 DEG C of incubations 30 minutes.
VI. discarding liquid in hole, dry, wash plate 5 times, method is with step IV.
VII. every hole adds substrate solution (TMB) 90 μ L, and ELISA Plate hatches about 15 minutes plus 37 DEG C of lucifuges of overlay film
(take the circumstances into consideration to shorten or extend according to reality colour developing situation, but may not exceed 30 minutes.When obvious gradient occurs in gauge orifice, i.e.
Can terminate).
VIII. every hole adds reaction terminating liquid 50 μ L, terminates reaction, and now blue standing turns yellow.The addition sequence of stop buffer
Should try one's best identical with the addition sequence of substrate solution.
Ⅸ. immediately with the microplate reader (the SPECTRA max plus384) optical density (OD in each hole of 450nm wavelength measurement
Value).
Ⅹ. substitute into equation according to the OD value of standard substance and calculate the detected value of serum sample FG γ, and judge itself and decision threshold
The magnitude relationship of value.
If FG γ detected value is more than or equal to decision threshold, then judge that patient has progressed to castration-resistant prostate cancer,
If FG γ detected value is less than decision threshold, judge that patient does not progresses to castration-resistant prostate cancer.Detect with FG γ in serum
Value is 19748.73mg/ml ~ 24137.33mg/ml, preferred 21943.03mg/ml is decision threshold.
Embodiment 1
The present embodiment is with FG γ detected value 21943.03mg/ml as judgment threshold, if FG γ detected value is more than or equal to
During 21943.03mg/ml, then judge that patient has progressed to castration-resistant prostate cancer, if FG γ detected value is less than
21943.03mg/ml then judge that patient does not progresses to castration-resistant prostate cancer.In the 86 example samples of the present embodiment, use
The sensitivity of test kit of the present invention diagnosis castration-resistant prostate cancer is 69%, and specificity is 86.4%.
The inspection value of the serum sample of the present embodiment is drawn in FIG, and two groups are compared P < 0.05, and difference has statistics meaning
Justice.
Fig. 2 is the ROC curve that FG γ diagnoses as castration-resistant prostate cancer, and abscissa is 1-specificity, vertical coordinate
For sensitivity, AUC=0.808, wherein ROC represents area under curve.It can be seen that sensitivity reaches 69%, specificity
Reach 86.4%.
Visible, use the present invention to detect the content of FG γ in serum of patients with prostate cancer, castration can be diagnosed rapidly and accurately
Repellence carcinoma of prostate, has higher sensitivity and specificity.
Claims (9)
1. the ELISA kit being used for detecting castration-resistant prostate cancer, it is characterised in that: described test kit includes FG
Detectable needed for gamma antibodies, and double antibody sandwich ELISA.
A kind of ELISA kit for detecting castration-resistant prostate cancer the most according to claim 1, its feature exists
In: described test kit includes being coated the ELISA ELISA Plate of FG gamma antibodies, standard substance, standard substance & sample diluting liquid, biotin mark
In the FG gamma antibodies of note, the horseradish peroxidase of Avidin labelling, cleaning mixture, substrate solution, reaction terminating liquid, overlay film one
Plant or several.
A kind of ELISA kit for detecting castration-resistant prostate cancer the most according to claim 1, its feature exists
In: detection sample is the serum of patients with prostate cancer.
4. for detecting a using method for the ELISA kit of castration-resistant prostate cancer described in claim 1-3, its
It is characterised by: comprise the following steps:
I. gather patients with prostate cancer whole blood sample, centrifuging and taking supernatant；
II. setting blank well, gauge orifice, testing sample hole respectively, blank well adds standard substance sample diluting liquid, remaining hole mark-on respectively
Quasi-product or testing sample, mixing；ELISA Plate adds overlay film, hatches；
III. discard liquid in hole, dry；Every hole adds biotin labeled FG gamma antibodies, and ELISA Plate adds overlay film, incubation；
IV. discard liquid in hole, dry, detersive enzyme target, then dry；
V. every hole adds the horseradish peroxidase of Avidin labelling, and ELISA Plate adds overlay film, incubation；
VI. discard liquid in hole, dry, detersive enzyme target, then dry；
VII. every hole adds substrate solution, and ELISA Plate adds overlay film, and lucifuge is hatched；
VIII. every hole adds reaction terminating liquid；
Ⅸ. measure the OD value in each hole under 450nm wavelength；
Ⅹ. calculate the detected value of serum sample FG γ according to the OD value of standard substance, and judge the magnitude relationship of itself and decision threshold.
The user of a kind of ELISA kit for detecting castration-resistant prostate cancer the most according to claim 4
Method, it is characterised in that: in described step I whole blood sample in room temperature place 0 ~ 4 hour or 0 ~ 8 DEG C overnight after in 500 ~ 1500 × g
Centrifugal 10 ~ 30 minutes.
The user of a kind of ELISA kit for detecting castration-resistant prostate cancer the most according to claim 4
Method, it is characterised in that: detersive enzyme target 1 ~ 5 time in described step IV, soak 1-2 minute every time；Detersive enzyme target 3 in step VI
~ 7 times, soak 1-2 minute every time.
The user of a kind of ELISA kit for detecting castration-resistant prostate cancer the most according to claim 6
Method, it is characterised in that: detersive enzyme target 3 times in described step IV, soak 1.5 minutes every time；Detersive enzyme target 5 in step VI
Secondary, soak 1.5 minutes every time.
The user of a kind of ELISA kit for detecting castration-resistant prostate cancer the most according to claim 4
Method, it is characterised in that: described decision threshold is 19748.73mg/ml ~ 24137.33mg/ml.
The user of a kind of ELISA kit for detecting castration-resistant prostate cancer the most according to claim 8
Method, it is characterised in that: described decision threshold is 21943.03mg/ml.
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| CN201610770860.2A CN106198985B (en) | 2016-08-31 | 2016-08-31 | For detecting the ELISA kit and application method of castration-resistant prostate cancer |
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| CN201610770860.2A CN106198985B (en) | 2016-08-31 | 2016-08-31 | For detecting the ELISA kit and application method of castration-resistant prostate cancer |
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| CN106198985A true CN106198985A (en) | 2016-12-07 |
| CN106198985B CN106198985B (en) | 2018-03-27 |
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| CN106198985B (en) | 2018-03-27 |
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