CN107151661A - A kind of people's excretion body protein, kit and its application - Google Patents
A kind of people's excretion body protein, kit and its application Download PDFInfo
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Abstract
The present invention relates to a kind of people's excretion body Dicer albumen and its application.Excretion body Dicer albumen of the present invention, is contained in blood plasma excretion body.The excretion body Dicer albumen can be applied as tumor markers.The present invention also provides the kit that can be used for detection blood plasma excretion body Dicer albumen.The kit of detection blood plasma excretion body Dicer albumen of the present invention, available for the CT of tumor patient (especially patients with lung cancer), aftertreatment recruitment evaluation and follow-up visit monitoring.
Description
Technical field
The invention belongs to medical biotechnology field, more particularly to a kind of excretion body Dicer albumen and its application, specifically
For a kind of ELISA (enzymes using excretion body Dicer albumen as tumor markers available for detection of early lung cancer and monitoring after operation
Linked immunosorbent assay) kit preparation and application.
Background technology
In recent years, lung cancer turns into one of major malignant tumor of threat human health, and its death rate occupies malignant tumour first place,
And the incidence of disease increases year by year.5 annual survival rates of lung cancer are only 13%, and 80% patient is dead in 1 year after diagnosis.The early stage of lung cancer
Diagnosis only 15%, but these patients 5 annual survival rates up to 60%~90%.Visible above, diagnosing, control lung cancer are pendulum
The urgent and difficult task in face of medical personnel.Clinically traditional method of lung cancer diagnosis has C-XF, bronchus
Spectroscopy, Phlegm Cells smear etc., because its symptom, sign are without specificity, with other PUD Ds, such as pneumonia, pulmonary tuberculosis etc.
Disease is difficult to differentiate.Because its specificity and sensitivity are limited, Most patients have been late periods when finding.Tumor markers is compared to it
He has higher specificity and sensitivity at traditional diagnosis method, is also increasingly used widely.
With the progress of oncological pathology, molecular biology research, research and the screening of tumor marker turn into lung
The study hotspot of cancer early diagnosis.Preferable tumor markers should be that sensitiveness is high, high specificity, expression quantity or blood plasma reclaimed water
It is flat with tumor tissues diffusion or Tumor size is into positive correlation.Clinical conventional lung cancer tumor mark includes at present：CEA（Cancer embryo
Antigen）、CYFRA21-1（The soluble fragments of Cyfra21-1）、SCC（Squamous cell carcinoma antigen）、NSE（Neuron-specific
Property enolase）And ProGRP（Gastrin-releasing peptide precursor）.CEA is a kind of with human embryos antigenic determinant
Acidoglycoprotein, it is a kind of tumor associated antigen, is present in kinds of tumors tissue, and it has higher examine in adenocarcinoma of lung
Disconnected value, when specificity is more than 90%, sensitiveness, up to 50%-70%, is also the one kind being clinically most widely used.Due to
Lung cancer morbidity mechanism is more complicated, and pathogenic molecular biology mechanism also shows diversity because of individual difference, and this also causes single
Molecular marked compound can not effectively indicate the early stage difference of lung cancer morbidity or the personal feature of prognosis.Consequently found that and developing
The tumor marker of Sensitivity and Specificity can be taken into account, applied to the clinical detection of lung cancer, to reach that lung cancer early stage examines
The mesh of disconnected, monitoring after operation and prognosis evaluation seems very necessary in the clinical practice of lung cancer.
Excretion body (Exosome) was found before 30 years by people.Excretion body is diameter about 30-150nm, and density exists
1.13-1.21g/mL vesicles.Excretion body is naturally occurring in body fluid, including blood, saliva, urine and breast milk, is living thin
The film vesica from late endosome (also referred to as many vesica bodies) of intracrine.The many of mother cell are carried in excretion body
Plant the important informations such as protein, lipid, DNA and RNA.And different histocytes source excretion bodies due to carrying protein not
Together, then different biological functions can be played.Excretion body plays pass by mutually melting with recipient cell during cell-cell communication
Key is acted on.Therefore albumen and RNA in these excretion bodies etc. is proved to have played huge tune for a variety of physiological events
Control is acted on, it is considered to be potential biomarker or therapeutic reagent.
Enzyme-linked immunosorbent assay (Enzyme-Linked ImmunoSorbent Assay, abbreviation ELISA) is
A kind of immunoenzyme technics grown up after immunofluorescence and radioimmunoassay technique.It is by known antigen or antibody suction
Surface of solid phase carriers is attached to, the technology that the antigen-antibody reaction for marking enzyme is carried out in solid phase surface.The technology can be used for detecting
Macromolecular antigen and specific antibody etc., have the advantages that quick, sensitive, easy, carrier is easy to standardization.Simultaneously as
ELISA technical conditions require low, easy to carry, normal to be occurred and easy commercialization, easy to operate and economy in the form of kit
Material benefit, it has also become a kind of to be most widely used and develop biological detection and analytical technology the most ripe.Based on the dual anti-of ELISA
Body sandwich method is the most common method for detecting antigen.
Dicer enzymes are a kind of endoribonucleases, belong to a member of specific recognition double-stranded RNA in RNase III families.
Dicer albumen has more domain, includes a DEXH boxes DBPA motif as being located at aminoterminal, and one
Positioned at the RNase motifs of c-terminus.It finds in drosophila at first, and shows on different organisms very high conservative
Property.The assignment of genes gene mapping of mankind's Dicer albumen is encoded in chromosome 14q32.13.Dicer albumen brain, heart, liver, lung,
Equal wide expression in the organs such as pancreas, kidney and placenta, and played a role in rnai pathway.It can with a kind of ATP according to
Bad mode progressively cuts the double-stranded RNA introduced including the various modes such as miRNA precursors, and RNA is degraded to 19-21bp's by cutting
There are 2 base protrusions at double-stranded RNA s (dsRNAs), 3 ' ends of each fragment.Single-stranded miRNA precursors can after Dicer is processed
Maturation is the miRNA that length is 20-24 nucleotides.Dicer albumen is located in the cytoplasm of mammal, and generally it is with calcium net
Albumen common location is in endoplasmic reticulum.The molecular size of Dicer albumen is 218kDa.
With research going deep into for excretion body and its secreted albumen and RNA etc., excretion body tumour generation, hair
Mechanism during exhibition, vicious transformation etc. is also more and more clearer.Research show tumour cell excretion body and normal cell it is outer
Secrete and have differences between body, the excretion of tumour knows from experience the growth and transfer that promote tumour, while the excretion body of tumors secrete can be with
Cell around changing, becomes tumour cell and promotes the growth of tumour.The research of inventor's early stage shows tumor patient
Ripe miRNA levels in excretion body substantially rise compared with normal person, at the same the miRNA precursors in same patient's excretion body then with
Time and constantly decline, this key protein required for having pointed out miRNA present in excretion body ripe.Some subsequent row are real
The conversion of the real cancer cell of checking depends on Dicer albumen, and tumour excretion body contains the corresponding protein such as Dicer, can processed
MiRNA precursors make its maturation be corresponding miRNA, so that the cell for changing near tumor cells is translated into tumour cell.
So, Dicer albumen can indicate the presence of tumour cell as a label, or be used as the target for the treatment of cancer.
Therefore, excretion body Dicer ELISA detection methods are set up, it is proposed that using excretion body Dicer as the tumour of mark
Detection architecture has important application value for clinical medicine detection of basic research and tumour etc..
The content of the invention
The invention provides a kind of excretion body Dicer albumen, depositing for Dicer albumen in excretion body is not only confirmed first
, and find expression of the secreting type Dicer albumen in normal person and tumor patient blood plasma there were significant differences, especially in lung
It is significantly raised in cancer patients with terminal blood plasma, and be in notable positive correlation with patients with lung cancer tumor load, therefore can be as a kind of preferable
Blood plasma label be used for patients with lung cancer CT, aftertreatment recruitment evaluation and follow-up visit monitoring, further provide
A kind of kit using excretion body Dicer albumen as novel tumor markers, is that the lung cancer of convenient quickly non-invasive is examined in vitro
It is disconnected to provide effective instrument.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：
A kind of excretion body Dicer albumen, the excretion body Dicer albumen is consistent with the Dier protein sequence features of intracellular
(GeneBank Shelf numbers are AAI50288.1), is contained in excretion body, and can be arrived in blood plasma excretion body by stable detection.
The present invention proves human plasma China and foreign countries by the way that Elisa technologies, Western-blot detections and qRT-PCR etc. are many-sided
The presence of body Dicer albumen is secreted, and determines that its size is 218KD, the Dicer albumen with intracellular is in the same size；In addition, in lung cancer
Inventor has found that excretion body Dicer protein concentrations are raised in patients blood plasma, and with very high Sensitivity and Specificity,
Excretion body Dicer albumen shows the activity related to tumour generation at many aspects, including promotes cell propagation and migrate, and resists
Apoptosis of tumor cells and medicine effect etc..
So far, inventor confirms excretion body Dicer albumen poor specificity opposite sex table in cancer patient's blood plasma first
Reach.
On the basis of above-mentioned technical proposal, further, above-mentioned excretion body Dicer albumen is used as tumor markers
Application.Application of the above-mentioned excretion body Dicer albumen as tumor markers in the preoperative detection of lung cancer and monitoring after operation.
Based on above-mentioned technical proposal, the present invention provides a kind of detection examination using excretion body Dicer albumen as tumor markers
Agent box, including：Goat anti human Dicer polyclonal antibodies, the detection antibody being coated with ELISA Plate are mouse anti human Dicer Dan Ke
The donkey anti-mouse IgG polyclonal antibodies and standard items of grand antibody, Streptavidin-horseradish peroxidase-labeled, the mark
Quasi- product are Dicer human recombination proteins, and the Dicer human recombination proteins are AAI50288.1 comprising NCBI GeneBank Shelf numbers
Shown amino acid sequence, it is preferable that the Dicer human recombination proteins are exactly to be by NCBI GeneBank Shelf numbers
Amino acid composition shown in AAI50288.1.
Using mentioned reagent box can be assisted by detecting the concentration of excretion body Dicer albumen in human plasma diagnosis and with
Patients with lung cancer is visited, another efficiently noninvasive instrument is provided for in-vitro diagnosis lung cancer.
Excretion body Dicer albumen of the present invention is equal to secreting type Dicer albumen, refers to be contained in blood plasma excretion body
Among, the method such as following kits stable detection can be included or quantitative Dicer albumen.
Specifically, mentioned reagent box of the present invention is the ELISA for detecting excretion body Dicer albumen in blood plasma
Kit, it is coated with a kind of Goat anti human Dicer polyclonal antibodies, passes through the excretion in coated Dicer antibody captures blood plasma
Body Dicer albumen, the content of excretion body Dicer albumen is detected with mouse anti human Dicer monoclonal antibodies.
The Cleaning Principle of kit of the present invention uses DAS-ELISA.
The detection object of kit of the present invention is the tumour of expression Dicer albumen in excretion body, the excretion body
Middle expression Dicer tumour includes lung cancer, breast cancer, cervical carcinoma, oophoroma, colorectal cancer and carcinoma of urinary bladder etc., and the tumour is excellent
Elect lung cancer as.
Kit of the present invention is used for early diagnosis, monitoring therapeuticing effect and the prognosis follow-up of lung cancer.
In kit of the present invention, the working concentration of the coated antibody is detection antibody described in 4ug/mL
Working concentration is 0.4ug/mL, and the working concentration of the enzyme-linked antibody is 1ug/mL.
Kit of the present invention has good specificity, sensitiveness and stability.
The specific advantage of kit of the present invention is：(1) a kind of new lung cancer molecular marker excretion body is introduced
Dicer albumen, by detecting the mark, can assist the clinical diagnosis of patients with lung cancer, determine whether patient's prognosis feelings
Condition, guiding treatment；Meanwhile, as an independent prognostic factor of lung cancer, the mark may also turn into treatment novel targets.(2)
The detection of the mark need to only draw blood and just can carry out, and be no intrusive mood liquid biopsy, and can detect at any time, be conducive to monitoring disease
Feelings.(3) ELISA detection techniques are in clinical practice comparative maturity, and simple and convenient, with low cost, suitable clinical detection.
Further, the coating is to Goat anti human's Dicer polyclonal antibodies on ELISA Plate, and mother liquid concentration is 0.2mg/
ML, the concentration of the working solution after 50 times of dilution is 4ug/mL；
1) detection antibody is mouse anti human Dicer monoclonal antibodies, and the concentration of mother liquor is 0.2mg/mL, utilizes biotin labeling
(biotin labeling reagent box is purchased from Japanese colleague, model LK03, and specific labeling method and implementation steps are said with reference to the product afterwards
Bright book), in use, the working solution concentration that 500 times of dilution is obtained is 0.4ug/mL；
2) the donkey anti-mouse IgG polyclonal antibodies of Streptavidin-horseradish peroxidase-labeled are (i.e.
Streptavidin-HRP IgG), the concentration of mother liquor is 0.5mg/mL；
3) standard items are Dicer human recombination proteins, and the Dicer human recombination proteins are comprising NCBI GeneBank Shelf numbers
Amino acid sequence shown in AAI50288.1, it is preferable that the Dicer human recombination proteins are exactly to be asked for by NCBI GeneBank
Number for the amino acid composition shown in AAI50288.1.It is synthesized by Nanjing Genscript Biotechnology Co., Ltd., its synthetic method
For the eukaryotic expression in sf9 insect cell and pass through affinity purification, the concentration of mother liquor is 100ng/uL.
Beneficial effect using above-mentioned further scheme is：
In kit of the present invention, the working concentration of the coated antibody is 4ug/mL, the work of the detection antibody
Concentration is 0.4ug/mL, and the working concentration of the enzyme-linked antibody is 1ug/mL, further ensures kit of the present invention
Convenience with good specificity, sensitiveness, stability and when using.
Further, in addition to coating buffer solution, cleaning solution, confining liquid, sample loading buffer, nitrite ion, reaction terminating liquid.
Further, the coating buffer solution is phosphate buffer (PBS), and its composition is 140mM NaCl, 2.7mM
KCl, 10mM Na2HPO4,1.8mM KH2PO4, pH value are 7.4；The cleaning solution is 0.05% to the addition of volume fraction
Tween-20 coating buffer solution；The confining liquid buffers for the coating for the bovine plasma albumin for being 1% containing volume fraction
Liquid；The composition of the sample loading buffer be 50mM Tris-HCl, 0.5M KCl, volume fraction be 1% bovine plasma albumin,
The Tween-20 of volume fraction 0.05%, pH value are 8.4；The composition of the reaction terminating liquid is 2M H2SO4.
Term used herein " working concentration " refers to the concentration of the used reagent such as antibody when being detected.
Kit of the present invention using secreting type Dicer albumen as tumor markers when in use, including following step
Suddenly：1. according to blood plasma working specification, separated plasma sample is collected, include plasma sample (including the lung of normal person and patient
The plasma sample of portion's benign lesion and patients with lung cancer)；2. the excretion body in separated plasma, extracts the total protein in excretion body；3.
Double-antibody method ELISA is tested, and is coated with by 96 hole elisa Plates polyclonal antibodies；Monoclonal antibody detects excretion body Dicer eggs
In vain；The priority step such as content of excretion body Dicer albumen in blood plasma is calculated to complete.
Preferably, the concrete operations of step 1 are：
1）The periphery whole blood of 2ml normal person or patients with lung cancer, 4 °C of storages or -80 °C of jellies are collected in vacuum blood collection tube
Deposit；
2）The whole blood frozen is in complete freeze thawing on ice, under the conditions of 4 °C, and 2000rpm is centrifuged 20 minutes；
3）Carefully the liquid of the bright clarification in upper strata is transferred in another clean centrifuge tube, proceed to next-step operation or-
80 °C freeze；
4）Clean environment is should be noted in above operating process, while needing wearing gloves and mouth mask etc., the peace of operating personnel is noted
Full operation.
Preferably, the concrete operations of step 2 are：
1）The isolation and purification of blood plasma excretion body
2）The blood plasma of complete freeze thawing state is centrifuged 20 minutes in 4 °C of lower 2000*g, removes cell and cell fragment；
3）Take supernatant to move to 16500*g under the conditions of new centrifuge tube, 4 °C to centrifuge 30 minutes, further remove cell fragment；
4）Take supernatant liquid to be transferred to centrifuge tube, add the phosphate buffer of 0.5 times of volume（PBS）, it is vortexed and mixes；
5）The Proteinase K of 0.05 times of blood plasma initial volume is added in the solution, it is preferable that the blood plasma of 500ul initial volumes needs
Add 25ul Proteinase Ks；
6）Sample is mixed, is incubated 10 minutes under the conditions of 37 °C；
7）Sample is passed through into 0.22um disposable filters, big vesica is removed, clear liquid is collected；
8）The clear liquid of collection is centrifuged 70 minutes under 110000*g, supernatant is abandoned, 5ml PBS solutions cleaning precipitation is added,
110000*g is centrifuged 70 minutes again, reject supernatant；
9）Solution is hanged with 100ul excretions body weight（PBS）All precipitations, -20 DEG C of preservations is resuspended.
10）The extraction of excretion body total protein
11）Excretion liquid solution and excretion body protein lysate are melted on ice；
12）Isometric excretion body protein lysate is added in excretion liquid solution after 25ul resuspensions, above and below pipettor
Pressure-vaccum mixes sample；
13）It is incubated 30 minutes on ice, sample is constantly mixed every 10 minutes reverse centrifuge tubes；
14）Under the conditions of 4 °C, 14,000*g centrifugations 10 minutes, transfer supernatant is placed on ice into clean centrifuge tube；
15）Measure protein concentration, it is preferable that wavelength 562nm extinction will be determined after 200 times of albumen Sample Dilution by BCA methods
Angle value, by standard curve that standard protein solution is drawn and its concentration that converts；
16）Albumen sample deposits in -80 °C, or is directly used in downstream analysis.
Preferably, the concrete operations of step 3 are：
1) Goat anti human's Dicer polyclonal antibodies that coating buffer solution (PBS) dilutes 50 times are added in ELISA Plate, per hole
100ul, 4 DEG C are coated with 24 hours；Liquid in hole is discarded, is patted dry on clean blotting paper, and washs with cleaning solution three times, often
Secondary 5 minutes；
2) 200uL confining liquids are added per hole, 37 DEG C are closed 2 hours；Liquid in hole is discarded, is patted dry on clean blotting paper,
And washed with cleaning solution three times, every time 5 minutes；
3) by standard items, (concentration is respectively 0ng/mL, 0.78125ng/mL, 1.5625ng/mL, 3.125ng/mL, 6.25ng/
ML, 12.5ng/mL, 25ng/mL, 50ng/mL) be separately added into step 4) processing hole in, per hole add 100uL, for making
Make standard curve；
4) testing protein sample is separately added into step 3) processing hole in, per hole add 100uL；Sealed membrane is capped, is placed in
37 DEG C are incubated 2 hours, for detecting testing sample；
5) by step 3) and step 4) liquid in hole discards, patted dry on clean blotting paper, and wash three with cleaning solution
It is secondary, 5 minutes every time；
6) to step 5) hole in, per hole be separately added into using sample loading buffer dilute 500 times biotin labeling mouse
Anti-human Dicer monoclonal antibodies (concentration is 0.4ug/mL), 100uL, 37 DEG C of reaction 2h are added per hole, sealed membrane is capped；Discard
Liquid in hole, is patted dry on clean blotting paper, and is washed three times, every time 5 minutes with cleaning solution；
7) 100uL Streptavidin-HRP IgG (concentration is 0.5mg/mL) are separately added into per hole, sealed membrane is capped,
37 DEG C of lucifuges react 20min；
8) 100uL nitrite ion TMB are added per hole, 10-30min is reacted at room temperature；
9) 50uL 2M H2SO4 terminating reactions are added per hole；
10) OD450 and OD 570 is determined in ELIASA to be worth；
11) the OD450-570 values (subtracting the numerical value that OD570 is measured using the OD450 numerical value surveyed) determined according to standard items,
Standard curve is drawn according to the value of measure using statistics mapping software, abscissa is excretion body Dicer albumen in testing sample
Concentration, ordinate be OD450-570 values；
12) excretion body Dicer albumen in measuring samples is calculated by the OD450-570 values of standard curve and unknown concentration sample
Concentration.
Brief description of the drawings
Fig. 1 is miRNA precursors in Plasma of The Patients With Lung Cancer excretion body and its maturation miRNA when being incubated at room temperature different time
Relative expression's situation.Figure 1A shows relative expression change of the miRNA precursors after being incubated 24 hours and 72 hours in excretion body
Situation；Figure 1B shows relative expression situations of change of the excretion cylinder mature miRNA after being incubated 24 hours and 72 hours.
Fig. 2 Westem Blotting methods detect Dicer albumen tables in patients with lung cancer and healthy individuals blood plasma excretion body
Up to situation.
Fig. 3 is the examination criteria curve of blood plasma excretion body Dicer albumen.
The sensitivity of Fig. 4 ELISA kits detection excretion body Dicer albumen and specificity.
Fig. 5 is the blood plasma excretion body that tumor patient, lung benign disease patient and healthy individuals are calculated according to formula
Dicer protein concentrations compare.
Embodiment
The principle and feature of the present invention are described below in conjunction with accompanying drawing, the given examples are served only to explain the present invention, not uses
In restriction the scope of the present invention.
The extraction for separating and its including total protein of excretion body in the collection of the plasma sample of embodiment 1, blood plasma
Inventor successively have collected three batches of patients with lung cancer during in May, 2015 to November in affiliated hospital of Wenzhou Medical University
（258, pathological characters are shown in Table 1）, lung's benign disease（41）And healthy individuals（110）Peripheral blood sample (difference batch
Collection, pretreatment, packing, preservation condition of secondary sample etc. are consistent), corresponding has been separated respectively by the method for centrifugation
The plasma sample of body.
The concrete operations of separation excretion body are from blood plasma
1）The blood plasma of complete freeze thawing state is centrifuged 20 minutes in 4 °C of lower 2000*g, removes cell and cell fragment；
2）Take supernatant to move to 16500*g under the conditions of new centrifuge tube, 4 °C to centrifuge 30 minutes, further remove cell fragment；
3）Take supernatant liquid to be transferred to centrifuge tube, add the phosphate buffer of 0.5 times of volume（PBS）, it is vortexed and mixes；
4）The Proteinase K Solution of 0.05 times of blood plasma initial volume is added in the solution, and the blood plasma of such as 500ul initial volumes is added
25ul Proteinase Ks；
5）Sample is mixed, is incubated 10 minutes under the conditions of 37 °C；
6）Sample is passed through into 0.22um disposable filters, big vesica is removed, clear liquid is collected；
7）The clear liquid of collection is centrifuged 70 minutes under 110000*g, supernatant is abandoned, 5ml PBS solutions cleaning precipitation is added,
110000*g is centrifuged 70 minutes again, reject supernatant；
8）Solution is hanged with 100ul excretions body weight（PBS）All precipitations, -20 DEG C of preservations is resuspended.
The operation of total protein is extracted from blood plasma excretion body：
1）Excretion liquid solution and excretion body protein lysate are melted on ice；
2）Isometric excretion body lysate is added in excretion liquid solution after 100ul resuspensions, passes through pressure-vaccum above and below pipettor
Mix sample；
3）It is incubated 30 minutes on ice, sample is constantly mixed every 10 minutes reverse centrifuge tubes；
4）Under the conditions of 4 °C, 14,000*g centrifugations 10 minutes, transfer supernatant is placed on ice into clean centrifuge tube；
5）Measure protein concentration, it is preferable that wavelength 562nm absorbance will be determined after 200 times of albumen Sample Dilution by BCA methods
Value, its concentration that converts by standard curve that standard protein solution is drawn；
6）Albumen sample deposits in -80 °C, or is directly used in downstream analysis.
The pathological classification of the patients with lung cancer of table 1
Embodiment 2：The expression quantity of the incubation at room temperature experiment of lung cancer blood plasma excretion body, detection miRNA and its precursor
With reference to embodiment 1 specific implementation method separated plasma in excretion body, by by the excretion of 3-5 pipes lung cancer or Healthy People
The body sample method that dispenses again of mixing obtains the excretion body sample of the matter such as equivalent.Then, healthy individuals and lung cancer is taken to suffer from respectively
The corresponding excretion body sample of person at ambient temperature without other any compounds in the case of place 24h and 72h respectively, with
Total serum IgE therein is extracted by TRIzol methods afterwards.
1ml TRIzol solution is added in 25ul excretion body weight suspensions（Invitrogen）, firmly room temperature after vortex oscillation
5 minutes are stood, 200ul chloroformic solutions are subsequently added, centrifuge tube is vibrated up and down to uniform state, is stored at room temperature after 3 minutes at 4 DEG C
Under the conditions of 12000rpm high speed centrifugations 15min.Upper strata aqueous phase is drawn into new centrifuge tube, isometric precooling isopropyl is added
Alcohol, is fully overturned after mixing, then stands 2 more than hour under the conditions of -20 DEG C.Under the conditions of 4 DEG C, 12000rpm high speed centrifugations
It is careful after 15min to discard supernatant, the 75% ethanol washing precipitation of 1ml precoolings is added, then 7500rpm is centrifuged at 4 DEG C
5min, discards liquid, 5min is placed at room temperature fully to dry precipitation, last 5ul adds the sterilized water without RNase and dissolved
Precipitation.RNA purity and concentration are measured using Nanodrop2000 ultraviolet specrophotometers, sample can be frozen in -80 DEG C.Carried
The total serum IgE taken passes through MultiScribe reverse transcriptase（Applied Biosystems）With oligo-d (T) and reverse transcription is
cDNA.Specifically, 150ng total serum IgE is by using primer as shown in table 2 and the steps of SuperScript III Platinum mono-
Method RT-qPCR kits（Invitrogen）Carry out the cDNA of reverse transcription miRNA precursors；And 10ng RNA is specific with containing
MiRNA primer TaqMan MicroRNA reverse transcription reagent box (Applied Biosystems) reagent is mixed, then according to manufacturer
Operation instruction complete reverse transcription.Reflection is incubated 30 minutes according to 16 °C above, and 42 °C are incubated 30 minutes, and 85 °C are incubated 5 points
The sequencing of clock is carried out.Real-time fluorescence quantitative PCR is in ABI Stepone Plus quantitative PCR apparatus (Applied
Biosystems on) carry out, using SYBR Green Master Mix (Applied Biosystems) and β-actin as
With reference to carrying out quantifying for miRNA precursors；It is used for using commercialized Assay-on-Demand (Applied Biosystems)
Each miRNA research.MiRNA expression is normalized by RNU6B expression.Each group all carries out three repetitions.
Purpose band is determined by melt curve analysis analysis and electrophoresis, Δ Δ Ct methods carry out relative quantification.In the excretion body of separate sources
The relative expression quantity of miRNA and its precursor under corresponding conditionses is as shown in Figure 1.
Excretion body is shown in the case of room temperature is placed 24 hours and placement 72 is small in Figure 1A, wherein miRNA precursors
Relative expression quantity situation of change.It can be seen that compared to the metastable expression of healthy individuals, in Plasma of The Patients With Lung Cancer excretion body
MiRNA precursors have obvious decline being incubated at room temperature after 72 hours.That Figure 1B is shown is then miRNA in excretion body
In relative expression's situation of different time points, the variation tendency with its precursor is on the contrary, corresponding maturation miRNA expression is being placed
Have after 72 hours and significantly lifted.Opposite variation tendency has also been pointed out potential from precursor to maturation in this
The process of miRNA conversions.
Table 2
| Target | Primer sequence |
| hsa-Actin | F: 5’CATGTACGTTGCTATCCAGGC3’ |
| hsa-Actin | R: 5’CTCCTTAATGTCACGCACGAT3’ |
| Pre-miR-10a,b | F: 5’TACCCTGTAGATCCGAATTTGTG3’ |
| Pre-miR-10a,b | R: 5’ATTCCCCTAGATACGAATTTGTGA3’ |
| Pre-miR-21 | F: 5’GCTTATCAGACTGATGTTGACTG3’ |
| Pre-miR-21 | R: 5’CAGCCCATCGACTGGTG3’ |
Embodiment 3：Verify the presence of blood plasma excretion body Dicer albumen
Step 1
According to the excretion body and total protein therein in the method separation patients with lung cancer and normal health subjects blood plasma in example 1,
And the absorption photometric value under 562nm wavelength is determined with BCA methods, concentration is calculated according to standard curve, protein quantification is that 30ug is rearmounted
It is standby in -80 DEG C, obtain the excretion body total protein in blood plasma source.
Step 2
The blake bottle culture ATCC for being 75cm with volume sources 293T cells, are changed into when cell reaches 70% degrees of fusion
10mL was without the culture medium of blood plasma 1640 (being purchased from GIBICO companies) culture 24 hours.5 bottles of upper honest and upright and thrifty 50mL are collected, using low
Warm freeze dryer be lyophilized into it is powdered after, add 5mL cell pyrolysis liquids (being century purchased from health, article No. is CW0889).After dissolving,
With BCA methods determine 562nm wavelength under absorption photometric value, according to standard curve calculate concentration, protein quantification be 30ug after be placed in-
80 DEG C standby, obtains cultivating the excretion body total protein of cell derived.
Step 3
According to the prompting culture cell of step 2, one bottle of cell being collected by centrifugation is taken, 1ml cell pyrolysis liquids, fully cracking is added
Afterwards, high speed centrifugation 10 minutes under the conditions of 4 °C, collect supernatant, are determined with BCA methods and calculate protein concentration, protein quantification is 30ug
After be placed in -80 DEG C it is standby, obtain the intracellular total protein of cell derived as positive control.
Step 4
The 30ug patients with lung cancer and healthy individuals blood plasma excretion body total protein sample that step 1 is obtained respectively, step 2 are obtained
30ug supernatants in excretion body total protein and the obtained 30ug intracellular total proteins of step 3 be respectively placed on 2 × SDS samples
In sample buffer solution, 100 DEG C, with time variation 5 minutes, are sequentially loaded onto in 10% SDS-PAGE gels, pass through 10% SDS-
PAGE is separated, and 0.2mm PVDF is gone to using Shi Shi electroporations (Bio-Rad) constant current 350mA ice bath transferring films 1h20min
Film, 37 DEG C of 5% skim milk of mass volume ratio is closed 1 hour, plus primary antibody polyclonal antibody anti-Dicer (by stoste according to
Volume ratio dilutes 400 times, and stoste is the polyclonal antibody purchased from SANTA CRUZ BIOTECHNOLOGY, INC., and article No. is
Sc-34601) it is incubated overnight；Plus (stoste is Donkey anti goat IgG-HRP to secondary antibody, and the concentration of working solution is by original
Liquid dilutes 1000 times according to volume ratio, purchased from proteintech, USA), it is incubated 1 hour at room temperature, chemiluminescence
(SuperSignal West pico Chemilu scent Substrate, 34080, Pierce), according to the explanation of kit
Book is developed, is fixed, and detects that excretion body total protein is (i.e. in human plasma respectively using above-mentioned Western Blotting methods
The albumen that step 1 is obtained) and culture people's 293T cells excretion body in Dicer albumen (albumen that i.e. step 2 is obtained) and
The result of intracellular Dicer albumen (albumen that i.e. step 3 is obtained) is shown in Fig. 2.
Embodiment 4 builds the detection kit using excretion body Dicer albumen as tumor markers
Kit using excretion body Dicer albumen as tumor markers is ELISA detection kits, including：The total excretion body of blood plasma
Re-suspension liquid, protein enzyme solution, excretion body lysate, coating to the Goat anti human Dicer polyclonal antibodies on ELISA Plate, detection
Antibody is mouse anti human Dicer monoclonal antibodies, the donkey anti-mouse IgG of Streptavidin-horseradish peroxidase-labeled is more
Clonal antibody, standard items, coating buffer solution, cleaning solution, confining liquid, sample loading buffer, nitrite ion, reaction terminating liquid.
1）The total excretion body weight suspension of blood plasma is phosphate buffer, and its composition is 140mM NaCl, 2.7mM KCl, 10mM
Na2HPO4、1.8mM KH2PO4, pH values are 7.4；
2）Protein enzyme solution is Proteinase K, and its concentration is 20 mg/mL；
3）Excretion body lysate, its composition is 50mM Tris-HCl (pH7.5), 150mM NaCl, the NP- of mass fraction 1%
40th, 5mM EDTA, 1mM sodium vanadates, 1mM PMSF, 10 μ g/mL Aprotinins, 10 μ g/mL leupeptins, sodium vanadate, PMSF, suppression peptide
Enzyme, leupeptin etc. are being added before use.
4) standard items are Dicer human recombination proteins, and its amino acid sequence is that NCBI GeneBank Shelf numbers are
Sequence shown in AAI50288.1, is synthesized by Nanjing Genscript Biotechnology Co., Ltd., and its synthetic method is in insect cell
Eukaryotic expression and pass through affinity purification in sf9, the concentration of mother liquor is 100ng/uL.
5) coating is to Goat anti human's Dicer polyclonal antibodies on ELISA Plate, purchased from SANTA CRUZ
BIOTECHNOLOGY, INC., catalog number (Cat.No.) are sc-34601, and mother liquid concentration is 0.2mg/mL, are diluted using PBS after 50 times
The concentration of working solution is 4ug/mL.
6) detection antibody is mouse anti human Dicer monoclonal antibodies, purchased from SANTA CRUZ BIOTECHNOLOGY,
INC., catalog number (Cat.No.) is sc-136981, and the concentration of mother liquor is 0.2mg/mL, after biotin labeling, in use, by biotin
It is 0.4ug/mL that the mouse anti human Dicer monoclonal antibodies of mark, which dilute 500 times of obtained working solution concentration,.
7) the donkey anti-mouse IgG polyclonal antibodies of Streptavidin-horseradish peroxidase-labeled are (i.e.
Streptavidin-HRP IgG), purchased from R＆D Systems, Catalog#DY998, the concentration of mother liquor is 0.5mg/mL.
8) coating buffer solution is phosphate buffer (i.e. PBS), and its composition is 140mM NaCl, 2.7mM KCl, 10mM
Na2HPO4,1.8mM KH2PO4, pH value are 7.4.
9) cleaning solution is the coating buffer solution that with the addition of the Tween-20 that volume fraction is 0.05%.
10) the coating buffer solution for the bovine plasma albumin that it is 1% containing volume fraction that confining liquid, which is,.
11) composition of sample loading buffer is that the ox blood slurry that 50mM Tris-HCl, 0.5M KCl, volume fraction are 1% is white
Albumen, volume fraction are 0.05% Tween-20, and pH values are 8.4.
12) nitrite ion TMB, purchased from R＆D Systems, Catalog#DY999.
13) composition of the reaction terminating liquid is 2M H2SO4.
Embodiment 5：The application method of excretion body Dicer protein detection kits
1) collect plasma sample and handled according to normal plasma separation method, obtain blood supernatant；
2) with reference to the excretion body separation in embodiment 1 and total protein extraction step, the total protein of blood plasma excretion body is obtained；
3) in 96 hole elisa Plates, it is 4ug/mL Goat anti human's Dicer polyclonal antibodies, 4 DEG C that 100uL concentration is added per hole
It is incubated 24h；Liquid in hole is discarded, is patted dry on clean blotting paper, is washed with cleaning solution three times, every time washing 5 minutes, filter paper
Blot；
4) 200uL confining liquids closing 2h (being closed under the conditions of 37 DEG C) is added per hole, liquid in hole is discarded, in clean blotting paper
On pat dry, washed with cleaning solution three times, every time washing 5 minutes, filter paper is blotted；
5) standard items or protein solution 100uL to be detected are added per hole, negative control hole is reserved plus PBS does blank control,
37 DEG C are incubated after 2h, discard liquid in hole, are patted dry on clean blotting paper, are washed with cleaning solution three times, every time 5 points of washing
Clock, filter paper is blotted；
6) 100uL mouse anti human Dicer monoclonal antibodies (biotinylation, concentration is 0.4ug/mL), 37 DEG C are added per hole
2h is incubated, is capped during incubation after the completion of sealed membrane, incubation and discards liquid in hole, patted dry on clean blotting paper, use cleaning solution
Wash three times, every time washing 5 minutes, filter paper is blotted；
7) 100uL Streptavidin-HRP IgG (concentration is 0.5mg/mL) are added per hole, sealed membrane, 37 DEG C of bars is capped
Lucifuge is reacted 20 minutes under part；
8) 100uL TMB colour developings are added per hole, are reacted at room temperature 20 minutes；
9) 50uL 2M H2SO4 terminating reactions are added per hole；
10) OD450 and OD570 values (450nm, 570nm, BIO-RAD Model) are determined in ELIASA.
The preparation of the standard curve of embodiment 6
By the step 4 of embodiment 5) add the standard items of various concentrations respectively, the concentration of standard items be respectively 0ng/mL,
0.78125ng/mL、1.5625ng/mL、3.125ng/mL、6.25ng/mL、12.5ng/mL、25ng/mL、50ng/mL.Remaining
The equal be the same as Example 5 of step.
The OD450-570 values determined according to standard items (subtract the number that OD 570 is measured using the numerical value surveyed of OD 450
Value), standard curve is drawn according to the value of measure using statistics mapping software, abscissa is excretion body Dicer in testing sample
The concentration of albumen, ordinate is OD450-570 values；The equation for measuring standard curve is：Y=0.0254x+0.0328, wherein,
Y is the absorbance (OD450-570 values) of sample well, and x is secreting type Dicer albumen absolute content (ng/mL) in blood plasma.Standard
Curve is as shown in Fig. 3, and R-Square (i.e. the regression coefficient of standard curve) is 0.999, represents that the kit is technically closed
Lattice.
According to excretion body Dicer protein contents in specific recognition blood plasma, judge whether patient suffers from pernicious lung cancer tumor.
Embodiment 7：Comparative example
In order to verify beneficial effect that the technical program possesses, while there is provided other conditions carry out contrast test.
Dicer human recombination proteins are used for standard items, its amino acid sequence is that NCBI GeneBank Shelf numbers are
Amino acid sequence shown in AAI50288.1, is synthesized by Nanjing Genscript Biotechnology Co., Ltd., using shown in embodiment 6
Method make standard curve.
Choose sensitive special pairing coated antibody and detection antibody verifies skill of the present invention using square formation titration
The beneficial effect of the testing conditions of art.Wherein coated antibody is Goat anti human's Dicer polyclonal antibodies, and detection antibody is biotin
Mouse anti human Dicer monoclonal antibodies after mark.
As shown in table 3, different coated antibody concentration and detection antibody concentration are set.
The concentration schematic diagram for the standard items that table 3 is added
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| A | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 |
| B | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 |
| C | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 |
| D | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
| E | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 | 3.125 |
| F | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 |
| G | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 | 12.5 |
| H | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 | 25 |
The setting of coated antibody concentration：
Coated antibody is diluted to 8ug/mL, 4ug/mL, 2ug/mL isoconcentration with coating buffer solution by row 96 orifice plates (96 of coating
Hole elisa Plates, Corning Incorporated, the U.S.), each concentration coating two is arranged, and two groups is set altogether, the numbering of the row at first group of place is 1-
6, the numbering of the row where second group is 7-12.
The setting of standard concentration：
In hole to numbering A1 to A12, numbering E1 to E12 Kong Zhongjun add weakly positive antigen (Dicer human recombination proteins
Concentration is 3.125ng/mL)；
In hole to numbering B1 to B12, numbering F1 to F12 Kong Zhongjun add weakly positive antigen (Dicer human recombination proteins
Concentration is 6.25ng/mL)；
In hole to numbering C1 to C12, numbering G1 to G12 Kong Zhongjun add strong positive antigen (Dicer human recombination proteins
Concentration is 12.5ng/mL)；
In hole to numbering D1 to D12, numbering H1 to H12 Kong Zhongjun add strong positive antigen (Dicer human recombination proteins
Concentration is 25ng/mL).
Detect the setting of antibody concentration：
Detection antibody is diluted to tetra- concentration of 400ng/mL, 200ng/mL, 100ng/mL, 50ng/mL with sample loading buffer,
It is separately added into above-mentioned hole.Wherein, numbering A1 adds concentration into C6, D1 to D6 hole to B6, C1 to A6, B1 and is
400ng/mL detects antibody；Numbering A7 adds concentration to B12, C7 to A12, B7 into C12, D7 to D12 hole
200ng/mL detects antibody；Numbering E1 adds concentration for 100ng/ to E6, F1 to F6, G1 into G6, H1 to H6 hole
ML detects antibody；Numbering E7 adds concentration for 400ng/mL to E12, F7 to F12, G7 to G12, H7 into H 12 hole
Detect antibody.
Plus substrate (TMB) colour developing, acid adding (sulfuric acid, concentration is 2M, and volume is 50uL) terminating reaction.Read and inhale respectively
Luminosity OD450-570 values (450nm, 570nm, BIO-RAD Model).
Remaining detailed specific steps (volume, the washing process of such as adding various reagents) is with reference to embodiment 5.
Experimental testing result, the working concentration for the coated antibody that the technical program is set and the work for detecting antibody
Make concentration, strong positive antigen liquid OD values are in 1.0-3.0 or so, negative reference OD values ＜ 0.2, far better than other detector bars
Part, meets the standard for building and being reached needed for kit.The working concentration for the coated antibody that the technical program is chosen is that 4ug/mL is
Best effort concentration, the technical program choose detection antibody working concentration be 0.4ug/mL be best effort concentration much
It is better than the Detection results of other concentration of setting.
Embodiment 8 utilizes blood plasma excretion body Dicer protein detection kits detection sample
Blood plasma is collected, routinely plasma separation method, 2000rpm centrifuges 20 points under the conditions of extracting 2mL vein peripheral bloods, 4 °C
Clock, suctions out upper plasma into clean centrifuge tube, -80 DEG C freeze, and melt on ice using preceding.Then according to described in embodiment 5
Detection method, Plasma of The Patients With Lung Cancer sample (n=258) is detected, and with standard items, normal individual plasma sample (n=
110) and lung's benign lesion (n=41) as control.Testing result progress is carried out using software SPSS 16.0 as follows
Statistical analysis.
The determination of Cutoff values：According to the sensitivity at each possible point of contact and specificity in statistical result, Youden is calculated
Index, and select its maximum point of contact to be critical point, determine in blood plasma content >=3.304ng/mL be on ROC curves most
Good critical point, i.e. Cutoff values (as shown in Fig. 4).The sensitivity of blood plasma excretion body Dicer protein content Diagnosis of malignant lung cancer
For 84.4%, specificity is 86.5%.
Compared according to formula y=0.0254x+0.0328 that embodiment 6 is obtained concentration calculated as shown in Fig. 5.
Concentration average (normal individual)=1.89ng/mL, concentration average (benign lesion)=2.65ng/mL, concentration average (suffer from by lung cancer
Person)=6.12ng/mL, is examined, excretion body Dicer albumen is in lung cancer using one-way analysis of variance (one-way ANOVA)
Expression is higher than lung's benign lesion and normal individual, with significant difference (Fig. 5).Excretion body Dicer albumen is in patients with lung cancer
And the differential expression in normal population blood plasma, alternatively, it is also possible to show the non-transfer group of excretion body Dicer albumen patients with lung cancer accordingly
And the expression after differential expression between transfer group and treatment in patients with lung cancer.Patients with lung cancer is divided into non-transfer group, transfer
Group and recurrence group, class mean=5.01ng/mL is not shifted, shifts class mean=6.23ng/mL, recurs class mean=6.74ng/mL,
Statistical analysis finds that Dicer concentration transfer group is higher than non-transfer group in lung cancer, and patients with recurrent excretion body Dicer expression is obvious high
In transfer group and non-transfer group (not illustrating).In addition, with the progress for the treatment of, Dicer concentration in patients blood plasma's excretion body
Gradually reduce, with significant difference, point out excretion body Dicer concentration to be likely to become the index of lung cancer therapy recruitment evaluation, not
After treatment group, chemotherapy after preoperative, postoperative, postoperative chemotherapy concentration average be respectively 8.39ng/mL, 5.94ng/mL, 4.38ng/mL,
2.98ng/mL。
Industrial applicibility
The kit that the present invention is provided is a kind of practicality kit available for clinical detection, by detecting patients blood plasma's excretion
In body Dicer level to assist the early diagnosis of lung cancer, prediction prognosis, to assess curative effect and follow-up visit monitoring swollen to early detection
Knurl recurs and shifted.The discovery of excretion body Dicer albumen, another point beyond blood plasma label is provided for pulmonary cancer diagnosis
Sub- label.Excretion body Dicer of the present invention sensitivity and specificity are higher.Therefore, the reagent of the present invention is utilized
Box, can combine imageological examination to carry out the preoperative early diagnosis of non-invasive lung cancer and monitoring after operation.And due to the biological marker
Thing is an independent prognostic factor, therefore can predict prognosis, the monitoring state of an illness, guiding treatment.
In view of the confirmation of result above and excretion body Dicer albumen, the present inventor further attempts to develop outside human plasma
Secrete detection kit-double crush syndrome kit of body protein.Experimental result confirms that kit is successfully constructed.The reagent
Box is 80.04% to the credible recall rate of patients with lung cancer, by the kit, the inventors discovered that normal individual and patients with lung cancer
There were significant differences for blood plasma Dicer contents.
Some terms and abbreviation in the present invention：mAb ：Monoclonal antibody, monoclonal antibody；pAb ：It is many
Clonal antibody, polyclonal antibody；BSA ：Bovine plasma albumin；Tween-20 ；IgG ：Immunoglobulin G；
TMB ：Tetramethyl benzidine；OD ：Absorbance；NP-40：Nonidet P40；PMSF：Phenylmethylsulfonyl fluoride.
It is important to note that instantiation and accompanying drawing are merely to explanation, it is clear that one of ordinary skill in the art
Various modifications and variations can be made to the present invention within the scope of the invention according to illustrating herein, these amendment and
Change is also included in the scope of the present invention.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Claims (10)
1. a kind of people's excretion body protein, it is characterised in that the excretion body protein is excretion body Dicer albumen.
2. a kind of excretion body Dicer albumen according to claim 1, it is characterised in that the Dicer albumen is located at excretion
In vivo and can be detected in human plasma.
3. a kind of excretion body Dicer albumen described in claim 1 or 2 is used as the application of tumor markers.
4. the excretion body Dicer albumen described in a kind of claim 3 is as tumor markers in the preoperative detection of lung cancer and postoperative prison
Application in survey.
5. a kind of kit for detecting excretion body Dicer albumen, it is characterised in that including the total excretion body weight suspension of blood plasma, albumen
Enzyme solutions, excretion body lysate.
6. according to claim 5 it is a kind of detect excretion body Dicer albumen kit, it is characterised in that
1）The total excretion body weight suspension of blood plasma is phosphate buffer, and its composition is 140mM NaCl, 2.7mM KCl, 10mM
Na2HPO4,1.8mM KH2PO4, pH value are 7.4；
2）The protein enzyme solution is Proteinase K, and its concentration is 20 mg/mL；
3）The excretion body lysate, its composition is 50mM Tris-HCl (pH7.5), 150mM NaCl, mass fraction 1%
NP-40,5mM EDTA, 1mM sodium vanadate, 1mM PMSF, 10 μ g/ml Aprotinins, 10 μ g/ml leupeptins, it is preferable that sodium vanadate,
PMSF, Aprotinin, leupeptin are being added before use.
7. a kind of kit for detecting excretion body Dicer albumen, it is characterised in that also including consisting of：It is coated with ELISA Plate
On Goat anti human Dicer polyclonal antibodies, detection antibody be mouse anti human Dicer monoclonal antibodies, Streptavidin-peppery
Donkey anti-mouse IgG polyclonal antibodies, the standard items of root peroxidase labelling, the standard items are Dicer human recombination proteins.
8. according to claim 7 it is a kind of detect excretion body Dicer albumen kit, it is characterised in that
1) coating is to Goat anti human's Dicer polyclonal antibodies on ELISA Plate, and mother liquid concentration is 0.2mg/mL, dilution 50
The concentration of working solution after times is 4ug/mL；
2) detection antibody is mouse anti human Dicer monoclonal antibodies, and the concentration of mother liquor is 0.2mg/mL, utilizes biotin labeling
Afterwards, in use, being by 500 times of obtained working solution concentration of mouse anti human Dicer monoclonal antibodies dilution of biotin labeling
0.4ug/mL ；
3) the donkey anti-mouse IgG polyclonal antibodies of Streptavidin-horseradish peroxidase-labeled, the concentration of mother liquor is
0.5mg/mL；
4) standard items, the concentration of mother liquor is 100ng/uL.
9. a kind of kit for detecting excretion body Dicer albumen according to claim 7 or 8, it is characterised in that also include
It is coated with buffer solution, cleaning solution, confining liquid, sample loading buffer, nitrite ion, reaction terminating liquid.
10. a kind of kit for detecting excretion body Dicer albumen according to claim 9, it is characterised in that
1) it is described coating buffer solution be phosphate buffer, its composition be 140mM NaCl, 2.7mM KCl, 10mM Na2HPO4,
1.8mM KH2PO4, pH value are 7.4；
2) cleaning solution is the coating buffer solution that with the addition of the Tween-20 that volume fraction is 0.05%；
3) the coating buffer solution for the bovine plasma albumin that it is 1% containing volume fraction that the confining liquid, which is,；
4) composition of the sample loading buffer is that the ox blood that 50mM Tris-HCl, 0.5M KCl, volume fraction are 1% starches white egg
In vain, volume fraction is 0.05% Tween-20, and pH values are 8.4；
5) composition of the reaction terminating liquid is 2M H2SO4.
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| CN112415206A (en) * | 2020-10-23 | 2021-02-26 | 上海良润生物医药科技有限公司 | Application of CD171 protein in exosome as tumor metastasis diagnosis marker |
| CN112415206B (en) * | 2020-10-23 | 2023-08-18 | 上海良润生物医药科技有限公司 | Application of CD171 protein in exosome as tumor metastasis diagnosis marker |
| CN115097143A (en) * | 2022-07-11 | 2022-09-23 | 东南大学 | Application of vitamin D binding protein in total exosomes of peripheral blood plasma in diagnosing depression |
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