CN107674884A - It is a kind of to be used to detect pancreatic cancer marker REG1A biotinylation liposome and its preparation method and application - Google Patents
It is a kind of to be used to detect pancreatic cancer marker REG1A biotinylation liposome and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of biotinylation liposome for being used to detect pancreatic cancer marker REG1A, it encapsulates reporter dna fragment, surface modification PEG2000 biotins；The present invention prepares the biotinylation liposome of parcel reporter dna fragment using DPPC, cholesterol, DSPE PEG2000, DSPE PEG2000 biotin film dispersion methods, and it is as immune labeled biology sensor.The invention further relates to a kind of method of the detection pancreas carcinoma marker REG1A based on above-mentioned biotinylation liposome, it includes：REG1A mouse resource monoclonal antibody coated elisa plates, REG1A albumen sample to be detected is added, reacted respectively with REG1A rabbits source polyclonal antibody, biotinylation goat-anti rabbit, Avidin again, it is eventually adding the optimized good biotinylation liposome of concentration, fluorescent quantitation LAMP amplifications are carried out, and the concentration of REG1A albumen is analyzed according to amplification.The present invention is combined using immune LAMP reactions with immunoliposome nano particle, can realize pancreas carcinoma marker REG1A high-sensitivity detection, and with the advantages of stability is good, cost is low, high specific.
Description
Technical field
The present invention relates to belong to nano biological detection technique field, more particularly to one kind for detecting pancreatic cancer marker
REG1A biotinylation liposome and its preparation method and application.
Background technology
Liposome (liposomes) is that phosphatide is dispersed in water a spheroidal, a part of aqueous phase of encapsulating to be formed
Vesicle.It usually contains one layer or multilayer immobilized artificial membrane, and big I is by 20nm to tens microns.The application of liposome is more
It is embodied in drug delivery, in-vitro transfection etc..With the development of liposome technology, liposome can encapsulate the same of a large amount of labels
When can combine various types of biological identification molecules, and there is higher surface area, interior aqueous phase volume is also bigger, can be by fat
Plastid is applied to immunoassay method, i.e. liposome immunization is analyzed.In operation, liposome can be promoted by surfactant
Rupture of membranes or the antibody of surface coupling start complement system etc., once completing, liposome membrane ruptures immunoassay immediately, release mark
Remember thing, so as to greatly significantly reduce the detection line of analyte, and liposome can wrap up signaling molecule, and this can be reduced
Non-specific adsorption.
Tumor markers characteristic is present in malignant cell, or is produced extremely by malignant cell, and it reflects
Tumorigenesis, lapse to, to the significant molecule of reaction treatment, the diagnosis and treatment to clinic play the role of important monitoring tumour.
Early diagnosis, early treatment, cancer mortality in examination crowd can be reduced, improve prognosis, improve life treatment.Thus, it is found that
Suitable and high sensitivity, specific detection tumor markers are current top priorities.With the continuous hair of Diagnostic Value of Several Serum Tumor Markers
Now with the detection of increasing low abundance proteinses, sensitivity and specificity for new protein detection method propose
Higher requirement.
Loop-mediated isothermal amplification technique (LAMP technology) is a kind of new nucleic acid amplification technologies, and it is in molecular Biological Detection
Field has very extensive application, has the characteristics that quick, efficient, high sensitivity and high specific.LAMP has many only
The advantages of special：(1) high sensitivity：LAMP reactions can be within 1 hour by the target gene amplification of several copies to 109Times；(2)
High specificity：LAMP technology uses four primers, can have with 6 different regions in identifying purpose sequence to target sequence
The selectivity of height, reduce the influence of non-target sequence；(3) without temperature thermal cycle：LAMP overall processes are at constant temperature (63 DEG C)
Under the conditions of can realize amplification；(4) special installation is not needed：LAMP method is simple to operate, in the application stage to instrument requirements
Very low, the thermostat of cheap and simple can be achieved with (such as thermostat water bath), can directly detect by an unaided eye white precipitate or
Green fluorescence, it can apply and be diagnosed with POCT, the application under adverse circumstances etc..Therefore, LAMP is combined with immunological technique, utilized
Nucleic acid amplification technologies amplified signal, in body fluid low content protide tumor markers carry out high sensitivity, it is noninvasive, quick,
Early diagnosis provides new method and thinking.
Cancer of pancreas is clinical common and higher grade malignancy disease.Counted according to latest data in 2014, its death
Number accounts for the 4th of malignant tumour, and second may be leapt to the year two thousand thirty.It is special to be additionally, since pancreas anatomical position, related neoplasms
Label poor specificity and early symptom unobvious, more than 80% cancer of pancreas are late period when finding, poor prognosis, are referred to as
" king of cancer ".The operative chance of Early pancreatic carcinoma is more than 50%, and postoperative 5 years survival rates are up to 80%.And middle and advanced stage pancreas
The chance that cancer undergos surgery is less than 20%, and postoperative 5 years survival rates are only 5%.Therefore, " early diagnosis " of cancer of pancreas is that disease is controlled
Treat and improve the key point of patient's survival rate.
The related tumor markers of pancreas includes sugar antigens (CA199), sugar antigens (CA242), carcinomebryonic antigen (CEA), pancreas
Adenoncus knurl fetal antigen (POA), pancreas specific antigen (PaA) etc..CA199 is carbohydrate albumen, and the sensitiveness of diagnosis of pancreatic cancer is
79%-81%, specificity is 82%-90%.But non-tumor disease such as chronic pancreatitis, cholelithiasis, diabetes etc. are often associated with
Low concentration increases, and threshold value of warning is high, the low shortcoming of specificity.2016, the report such as Crnogorac-Jurcevic passed through detection
Three kinds of biomarker protein REG1A, LYVE1 and TFF1 contents in urine, carry out joint early diagnosis, accuracy rate can to cancer of pancreas
Up to 90%, provide new approaches for the diagnosis and treatment of clinic.REG1A albumen be a kind of islet cells endogenous growth because
Son, mainly express, formed to gastronintestinal system inflammatory disease and tumour related in pancreas.Therefore, excavate highly sensitive and specific
Detection urine in this mark REG1A albumen new method, the early diagnosis to ductal pancreatic adenocarcinoma is significant.
The content of the invention
The defects of it is an object of the invention to overcome in the prior art, detected based on liposomal encapsulated DNA hypersensitivitys
The structure of low-abundance protein, the present invention propose it is a kind of be used for detect pancreatic cancer marker REG1A biotinylation liposome and
Its preparation method, and propose that one kind is directed to cancer of pancreas urine Specific marker REG1A high-sensitivity detecting methods, for detection
The concentration of REG1A albumen, so as to carry out the early detection of cancer of pancreas, recurrence and transfer detection indirectly.
To achieve the above object, the present invention adopts the following technical scheme that：
First purpose of the present invention is to provide a kind of biotinylation lipid for being used to detect pancreatic cancer marker REG1A
Body, the liposomal encapsulated reporter dna fragment of biotinylation, its surface modification PEG2000- biotins.
Further, the biotinylation liposome size is 150 ± 30nm.
Second object of the present invention is to provide a kind of preparation method of above-mentioned biotinylation liposome, and this method uses
DPPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-biotin film dispersion method prepare the life of encapsulating reporter dna fragment
Thing elementization liposome.
In order to further optimize the preparation method of above-mentioned biotinylation liposome, the technical measures that the present invention is taken also are wrapped
Include：
Further, the preparation method of above-mentioned biotinylation liposome comprises the following specific steps that：
A) DPPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-biotin for weighing scheduled volume are positioned over centrifugation
Guan Zhong, add the chloroform and methanol solvate of scheduled volume, ultrasonic dissolution；
B) solution after step a) ultrasounds is moved into flask to be evaporated under reduced pressure, lipid film is formed in the inwall of flask；
C) the reporter dna fragment solution being pre-configured with is added dropwise into inwall in step b) to be formed in the flask of lipid film, together
When water-bath and rock flask, dissolve the lipid film on inwall and depart from, obtain multilamellar liposome；
D) multilamellar liposome of encapsulating reporter dna fragment in step c) is put into water bath sonicator instrument while carries out ice-water bath
And ultrasound；
E) by the solution centrifugal after step d) ultrasounds, not scattered, larger liposome is removed；Aspirate supernatant is dialysed, and is gone
Except the DNA molecular not embedded, biotinylation liposome is obtained；
F) the biotinylation liposome Cord blood for obtaining step e).
Further, in the step a), DPPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-biotin
The ratio between mole is (80~120)：(80~120)：(5~15)：The volume ratio of (0.5~2), chloroform and methanol is (3~9)：
(0.5~2)；Further, the ratio between DPPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-biotin mole
For 95：95：9：1, the volume ratio of chloroform and methanol is 6：1.
Further, 5~30min of ultrasonic dissolution in the step a)；Further it is 10min.
Further, the temperature that operation is evaporated under reduced pressure is 25~45 DEG C, is further 35 DEG C.
Further, centrifugally operated is 3000rpm 15min in the step e), and dialysis uses 300KD bag filters.
Further, the biotinylation liposome size that the step e) is obtained is 150 ± 30nm.
Further, the Cord blood temperature that the step f) is obtained is 0~10 DEG C；Further it is 4 DEG C.
Third object of the present invention is to provide a kind of detection pancreatic cancer marker based on above-mentioned biotinylation liposome
REG1A method, this method are used for the non-diagnostic and purpose of non-treatment, and it comprises the following steps：
1) preparation of biotinylation liposome；
2) REG1A mouse resource monoclonal antibody coated elisa plate, REG1A albumen sample to be detected is added, then respectively with
REG1A rabbits source polyclonal antibody, biotinylation goat anti-rabbit antibody, Avidin reaction；
3) the biotinylation liposome of predetermined concentration prepared by step 1) is added, fluorescent quantitation LAMP amplifications are carried out after rupture of membranes
Reaction；
4) according to the concentration of the amplified reaction interpretation of result REG1A albumen of step 3).
In order to further optimize above-mentioned detection pancreatic cancer marker REG1A method, the technical measures that the present invention is taken
Also include：
Further, the preparation of step 1) the biotinylation liposome uses the hereinbefore preparation method having been mentioned.
Further, the concentration of the biotin liposome is 0.12-0.24mg lipid/ml.
Further, the rupture of membranes step of step 3) uses rupture of membranes liquid, and the rupture of membranes liquid includes Triton X-100；More enter one
Step ground, the rupture of membranes liquid are 10Mm the Triton X-100, PH=9.0 in 10mM boric acid.
Further, in above-mentioned detection method, antibody coating concentration in REG1A monoclonal rats source is 1 μ g/ml, REG1A rabbits
Source polyclonal antibody detectable concentration is 1 μ g/ml, and biotinylation goat anti-rabbit antibody concentration is 1：1000, Avidin uses dense
Spend for 2 μ g/ml, the concentration of biotinylation liposome is 0.20mg lipid/ml.
Further, in above-mentioned detection method, REG1A monoclonal rats source antibody is 0.2mg/ml Abcam
AB201643, REG1A rabbit source polyclonal antibody are 1mg/ml Abcam AB47099.
Further, the minimum detected value of REG1A albumen is 1fg/ml in this method.
Further, analysis method is specific as follows in the step 3)：Using amplification corresponding to REG1A standard concentration values
CT values draw standard curve, and the CT value establishing criteria curves that REG1A albumen pattern detections to be detected obtain are analyzed, from
And obtain the concentration of REG1A albumen.
The invention provides REG1A albumen in a kind of noninvasive high-sensitivity detection urine, can (indirect) realize cancer of pancreas
Early diagnosis, provide new approaches for clinic diagnosis.This method design mainly includes following components：(1) reporter dna fragment
Synthesis and inner primer, outer primer (primer adds in last reaction system, is added together with reaction solution etc.) design；(2) fat
The synthesis of plastid and reporter dna parcel, while determine envelop rate, particle size, retention volume, phospholipid concentration and Electronic Speculum sign etc.
Deng；(3) sandwich immune response；(4) LAMP is expanded after liposome rupture of membranes, is characterized by the change of fluorescence signal CT values
The concentration of albumen is captured, so as to the concentration of REG1A albumen in accurate overdelicate analysis urine.
The present invention devises the tumor markers REG1A of cancer of pancreas detection method based on following principle：Three generations's liposome
DNA is encapsulated as report molecule, surface is PEG2000- biotins, passes through life with the sandwich system of REG1A antigen-antibodies
Thing element-Avidin-Biotin is connected.After rupture of membranes, the signal that DNA reporter molecule is carried out by fluorescent quantitation LAMP amplifies, CT values
Change can with indirect reaction REG1A antigenic content, and with this do standard curve detection urine in REG1A albumen.In addition,
Contrasting other protein molecular analysis results, to show that this method possesses superior specific and selective, while to the urine sample of complexity
This method displays that preferable stability when this is analyzed, and the diagnosis of cancer of pancreas can be carried out with extreme early, thus possesses preferably
Application prospect.
The present invention uses above-mentioned technical proposal, compared with prior art, the invention has the advantages that：
Fluorescence analysis platform of the present invention based on LAMP technology structure liposome DNA reporter molecule, can be with highly sensitive
Degree, super low-abundance detection pancreas carcinoma marker REG1A albumen, this method is easy to operate, is carried out in original ELISA disengaging
The amplification of signal three times, by fluorescence LAMP quantitative analysis of protein contents, it can realize that pancreas carcinoma marker REG1A's is highly sensitive
Degree detection, and with the advantages of stability is good, cost is low, high specific.
The present invention is combined using immune LAMP reactions with immunoliposome nano particle, report nucleic acid fragment is carried out double
The amplification of weight signal, further improves sensitivity and specificity.Combining diagnosis detects Early pancreatic carcinoma urinary biomarkers albumen
REG1A contents, new approaches are provided for the diagnosis and treatment of clinic.
Its advantage and feature are specific as follows：(1) from nucleic acid molecules level, ground using nucleic acid amplification technologies are highly sensitive
Study carefully biomarker protein content in urine, using nucleic acid amplification technologies high-sensitivity detection biomarker protein matter, for
Cancer of pancreas early stage, the biomarker protein diagnosis detection of hypersensitive, high specific；(2) DNA parcel fat is innovatively combined
Plastid, it can also can be combined with amplification detection signal with nucleic acid amplification technologies, so as to indirect detection biomarker protein
Matter；(3) present invention integrates immune LAMP nucleic acid amplification technologies and DNA parcel liposome technology, builds three kinds of biology marks in urine
The special high-sensitivity detection new system of will thing protein, research Early pancreatic carcinoma diagnosis new system, have pole in clinical practice
Big is perspective and innovative.
Brief description of the drawings
Fig. 1 is the schematic flow sheet based on liposomal encapsulated DNA hypersensitivitys detection REG1A protein determinations；
Fig. 2 is the amplification schematic diagram of the concentration optimization of biotinylation liposome；Wherein part A biotinylation fat
The concentration of plastid is 1.2mg/ml, and the concentration of part B biotinylation liposome is 0.24mg/ml, C portion biotin
The concentration for changing liposome is 0.12mg/ml, and the concentration of D part biological elementization liposomes is 0.06mg/ml；
Fig. 3 is in the amplification figure and solution detected based on biotinylation liposome to various concentrations recombinant protein
Linear relationship chart between REG1A concentration and fluorescence LAMP CT numerical value.
Fig. 4 is as control, detection with 5 kinds of different albumen (P-gp, MUC1, IL-6, cattle mucins, BSA)
The specific result figure of REG1A albumen.
Embodiment
The invention provides a kind of biotinylation liposome for being used to detect pancreatic cancer marker REG1A, it encapsulates report
DNA fragmentation, its surface modification PEG2000- biotins；The present invention also provides a kind of preparation side of above-mentioned biotinylation liposome
Method, and a kind of method of the detection pancreatic cancer marker REG1A based on above-mentioned biotinylation liposome is provided.
With reference to the accompanying drawings and examples, the embodiment of the present invention is further described.Following examples are only
For clearly illustrating technical scheme, and can not be limited the scope of the invention with this.
Embodiment 1
The present embodiment is that it includes for the preparation method for the biotinylation liposome for detecting pancreatic cancer marker REG1A
Following steps：
A) assay balance weighing DPPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-biotin (rub between four
The ratio between your amount is 95：95：9：1, gross mass 36.5mg), place in 10ml centrifuge tubes, add 3.43ml chloroforms and 571 μ l first
(the two volume ratio is 6 to alcoholic solvent：1, cumulative volume 4mL), ultrasonic 10min accelerates dissolving.
B) aforesaid liquid after ultrasound adds 50ml eggplant-shape bottles, is put into the rotary evaporation instrument put up, and depressurizes lower evaporate
Organic solvent (35 DEG C), one layer of thin lipid film is formed in flask inwall.
C) pre-configured 2ml PBS are added dropwise in flask containing 1 μ g DNA solutions, while spend in water-bath and shake at 45 DEG C
Flask is shaken, departs from adipose membrane dissolving on inwall, now obtains multilamellar liposome.
D) liposome for encapsulating DNA is put into ice-water bath in water bath sonicator instrument, 4min ultrasounds -1min stops (20W) circulation 10
It is secondary.
E) solution centrifugal 3000rpm 15min are removed as scattered, larger liposome, saturating with 300KD after Aspirate supernatant
Bag is analysed, removes the DNA molecular not embedded.
F) liposome puts 4 DEG C of preservations.
The above-mentioned liposomal encapsulated reporter dna fragment of biotinylation, its surface modification PEG2000- biotins, the biotinylation
The size of liposome is 150 ± 30nm.
Embodiment 2
The present embodiment is to be used for the non-diagnostic and detection cancer of pancreas of non-treatment purpose based on above-mentioned biotinylation liposome
Label REG1A method, based on liposomal encapsulated DNA hypersensitivitys detection REG1A protein determinations schematic flow sheet such as Fig. 1
It is shown.
Comprise the following steps that：
1) the μ g/ml of REG1A monoclonal rats source antibody 1 are coated with the healthy and free from worry ELISA Plates of coast 9018, and 4 DEG C overnight.
2) PBST (0.05%Tween 20PH7.4) board-washing 2 times, the μ l of detection plate 23%BSA 250 are closed into 1h.Repetition is washed
Plate 4 times.
3) will be added to after 10 times of dilutions of recombinant protein in detection plate, 37 DEG C of incubation 1h.Repeat board-washing 4 times.
4) polyclonal mouse source antibody 1 the μ g/ml, 37 DEG C of incubation 1h of REG1A is added.Repeat board-washing 4 times.
5) 1 is added：1000 biotinylation goat anti-rabbit antibody each 100 μ l, 37 DEG C of incubation 1h.Repeat board-washing 4 times.
6) 100 μ l Avidins 2 μ g/mL, 37 DEG C of incubation 1h are added.Repeat board-washing 4 times.
7) 300 μ l blank SUV (0.2mg lipid/ml) closings, 37 DEG C of incubation 1h are added.Repeat board-washing 4 times.
8) liposome prepared (is prepared) into dilution using the method described in embodiment 1 to be added in coated hole
(0.2mg lipid/ml), 37 DEG C of incubation 1h.Repeat board-washing 4 times.
9) by 100 μ l DNase I (100IU) (10mM CaCl2, 10mM MgCl2, 20mM HEPES, PH7.8) add it is micro-
Kong Zhong, 37 DEG C of 30min, 80 DEG C of 10min.PBS is washed 5 times.
10) 25 degrees Celsius of 100 μ l ruptures of membranes liquid (10Mm Triton X-100, PH 9.0 in 10mM boric acid) are added to incubate
Educate 15min.
11) fluorescent quantitation LAMP determines (25 μ L) and carries out the concentration analysis of REG1A albumen.
In the embodiment, used antibody and recombinant protein are as follows：REG1A monoclonal rats source antibody is 0.2mg/ml
Abcam AB201643, REG1A multi-clone rabbits source antibody is that 1mg/ml Abcam AB47099, REG1A recombinant protein are 10 μ g
Abcam AB73778。
Embodiment 3
The present embodiment is the optimization of the concentration of biotinylation liposome, and its step is same as Example 2, the present embodiment
The concentration of the biotinylation liposome of use is respectively that 1.2mg/ml, 0.24mg/ml, 0.12mg/ml, 0.06mg/ml (divide
Part A that Dui Yingyu be in Fig. 2, part B, C portion, D parts), from Fig. 2 amplification, when biotinylation liposome
Concentration when being 1.2mg/ml, there is " S " type amplification curve in REG1A protein groups and blank control group；Work as biotinylation
When the concentration of liposome is 0.24 or 0.12mg/ml, only there is " S " type amplification curve in REG1A protein groups；Work as biotinylation
When the concentration of liposome is 0.06mg/ml, each experimental group does not occur " S " type amplification curve.
From above-mentioned experimental result, in detection pancreatic cancer marker REG1A of the present invention method, biotin
The preferable concentration for changing liposome is 0.12-0.24mg lipid/ml.
Embodiment 4
The present embodiment is detected based on biotinylation liposome to various concentrations recombinant protein, to determine minimum detection limit
Value, and standard curve is drawn using amplification CT values corresponding to REG1A standard concentrations value.
The concentration for the recombinant protein that the present embodiment uses includes 1 μ g/ml, 100ng/ml, 1ng/ml, 10pg/ml, 100fg/
ml、1fg/ml、10ag/ml.Its operating procedure is same as Example 2.Its final result as shown in figure 3, with the increase of concentration,
The increase of CT values.It can confirm that detection method of the present invention is minimum in from the above to can be achieved to 1fg/ml Sensitive Detections,
And the result also show the good linear relation between REG1A concentration and fluorescence LAMP CT numerical value, result above shows this
A series of detection means can realize the detection of REG1A albumen ultra-high sensitives.
Embodiment 5
The present embodiment demonstrates the specificity of detection REG1A albumen.
To detect specificity of the experimental method to pancreas carcinoma marker REG1A, clinical normal Check-up crowd urine is collected,
Exclude organic disease.It is separately added into REG1A specific recombinant proteins 500ng/ml, 100ng/ml, 1ng/ml.Using above-mentioned life
Thing elementization liposome-LAMP method detection, detected value and addition standard volume are contrasted.It the results are shown in Table 1
The detection of recombinant protein is added in the urine of table 1
In addition, the present embodiment is used with other different albumen (P-gp, MUC1, IL-6, cattle mucins, BSA) in 5
It is respectively that 100ng/ml disturbs albumen and REG1A albumen to enter to concentration according to the operating method described in embodiment 2 as control
Row colorimetric detection.Its result is as shown in figure 4, when containing 100ng/ml REG1A albumen in solution, final fluorescence intensity highest.
And result is all relatively and very big with REG1A albumen result difference during containing other interference albumen, illustrate this method for REG1A
Albumen has preferably selectivity.
It is of the present invention to be used for non-diagnostic and non-control based on above-mentioned biotinylation liposome from above-described embodiment
The detection pancreatic cancer marker REG1A for the treatment of purpose method, have for detection REG1A albumen selective and special well
Property, design and operating process are easy, cost is cheap, hypersensitivity, thus following to cancer of pancreas early diagnosis and low abundance
Protein Detection has very big potential using value.
The specific embodiment of the present invention is described in detail above, but it is only used as example, and the present invention is not intended to limit
In particular embodiments described above.To those skilled in the art, it is any to the practicality carry out equivalent modifications and replace
In generation, is also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and repair
Change, all should be contained within the scope of the invention.
Claims (9)
- A kind of 1. biotinylation liposome for being used to detect pancreatic cancer marker REG1A, it is characterised in that the biotinylation fat Plastid encapsulates reporter dna fragment, its surface modification PEG2000- biotins.
- 2. a kind of biotinylation liposome for being used to detect pancreatic cancer marker REG1A according to claim 1, its feature It is, the biotinylation liposome size is 150 ± 30nm.
- A kind of 3. preparation as claimed in claim 1 or 2 for being used to detect pancreatic cancer marker REG1A biotinylation liposome Method, it is characterised in that the preparation method is thin using DPPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-biotin Film dispersion method prepares the biotinylation liposome of encapsulating reporter dna fragment.
- 4. the preparation method according to claim 3 for being used to detect pancreatic cancer marker REG1A biotinylation liposome, Characterized in that, the preparation method comprises the following steps：A) DPPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-biotin for weighing scheduled volume are positioned in centrifuge tube, Add the chloroform and methanol solvate of scheduled volume, ultrasonic dissolution；B) solution after step a) ultrasounds is moved into flask to be evaporated under reduced pressure, lipid film is formed in the inwall of flask；C) the reporter dna fragment solution being pre-configured with is added dropwise into inwall in step b) to be formed in the flask of lipid film, while water Bathe and rock flask, dissolve the lipid film on inwall and depart from, obtain multilamellar liposome；D) multilamellar liposome of encapsulating reporter dna fragment in step c) is put into water bath sonicator instrument while carries out ice-water bath and surpass Sound；E) by the solution centrifugal after step d) ultrasounds, not scattered, larger liposome is removed；Aspirate supernatant is dialysed, and is removed not The DNA molecular of embedding, obtain biotinylation liposome；F) the biotinylation liposome Cord blood for obtaining step e).
- 5. the preparation method according to claim 4 for being used to detect pancreatic cancer marker REG1A biotinylation liposome, Characterized in that, in the step a), DPPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-biotin mole The ratio between be (80~120)：(80~120)：(5~15)：The volume ratio of (0.5~2), chloroform and methanol is (3~9)：(0.5~ 2)。
- 6. a kind of detection pancreatic cancer marker REG1A of biotinylation liposome based on described in claim 1 or 2 method, It is characterised in that it includes following steps：1) preparation of biotinylation liposome；2) REG1A mouse resource monoclonal antibody coated elisa plate, REG1A albumen sample to be detected is added, then respectively with REG1A Rabbit source polyclonal antibody, biotinylation goat anti-rabbit antibody, Avidin reaction；3) the biotinylation liposome of predetermined concentration prepared by step 1) is added, it is anti-that fluorescent quantitation LAMP amplifications are carried out after rupture of membranes Should；4) according to the concentration of the amplified reaction interpretation of result REG1A albumen of step 3).
- 7. detection pancreatic cancer marker REG1A according to claim 6 method, it is characterised in that the biotinylation The concentration of liposome is 0.12-0.24mg lipid/ml.
- 8. detection pancreatic cancer marker REG1A according to claim 6 method, it is characterised in that the rupture of membranes of step 3) Step uses rupture of membranes liquid, and the rupture of membranes liquid includes Triton X-100.
- 9. detection pancreatic cancer marker REG1A according to claim 6 method, it is characterised in that REG1A in this method The minimum detected value of albumen is 1fg/ml.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113684245A (en) * | 2020-05-19 | 2021-11-23 | 南方医科大学第五附属医院 | Detection kit and detection method for MPT64 protein |
| WO2023284579A1 (en) * | 2021-07-14 | 2023-01-19 | 深圳市盛波尔生命科学技术有限责任公司 | ANTIBODY COMBINATION AGAINST REGENERATING ISLET-DERIVED PROTEIN 1α AND DETECTION KIT COMPRISING SAME |
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| WO2016124947A1 (en) * | 2015-02-05 | 2016-08-11 | Queen Mary University Of London | Biomarkers for pancreatic cancer |
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| WO2016124947A1 (en) * | 2015-02-05 | 2016-08-11 | Queen Mary University Of London | Biomarkers for pancreatic cancer |
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| JUNKUN HE ET AL.: "Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system", 《J NANOBIOTECHNOLOGY.》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113684245A (en) * | 2020-05-19 | 2021-11-23 | 南方医科大学第五附属医院 | Detection kit and detection method for MPT64 protein |
| WO2023284579A1 (en) * | 2021-07-14 | 2023-01-19 | 深圳市盛波尔生命科学技术有限责任公司 | ANTIBODY COMBINATION AGAINST REGENERATING ISLET-DERIVED PROTEIN 1α AND DETECTION KIT COMPRISING SAME |
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