CN108414755A - Protein chip that is a kind of while detecting four bladder carcinoma markers in urine - Google Patents
Protein chip that is a kind of while detecting four bladder carcinoma markers in urine Download PDFInfo
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- CN108414755A CN108414755A CN201711007608.7A CN201711007608A CN108414755A CN 108414755 A CN108414755 A CN 108414755A CN 201711007608 A CN201711007608 A CN 201711007608A CN 108414755 A CN108414755 A CN 108414755A
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- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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Abstract
Protein chip a kind of while that detect four bladder carcinoma markers in urine of the present invention belongs to and is tested by means of measuring the chemically or physically property of material or analysis of material technical field.The protein chip is the protein chip containing NMP22 microballoons, BTA microballoons, CD20 microballoons and TERT microballoons simultaneously；NMP22 monoclonal antibodies, BTA monoclonal antibodies, CD20 monoclonal antibodies and the TERT monoclonal antibodies that the NMP22 microballoons, BTA microballoons, CD20 microballoons and TERT microballoons are coated with microballoon and are coupled respectively by the colloidal gold for being embedded in quantum dot form.The present invention has the following advantages：It can be directed to specificity high NMP22, BTA, CD20 and TERT simultaneously, the early detection and recurrence for carrying out carcinoma of urinary bladder detect；Chip detection means is quick, time saving and energy saving, is suitble to wide clinical application.
Description
Technical field
The invention belongs to be tested or analysis of material technical field by means of measuring the chemically or physically property of material, specifically
It is related to protein chip that is a kind of while detecting four bladder carcinoma markers in urine.
Background technology
Carcinoma of urinary bladder is the common malignant tumour of urinary system, and the incidence of China's bladder carcinoma in males has rising year by year and youth
The trend of change.Although the diagnosing and treating technology of carcinoma of urinary bladder is greatly improved in recent years, the death rate of carcinoma of urinary bladder still occupies
It is high not under.Carcinoma of urinary bladder lacks effective therapy target and postoperative assessments index at present, and about 5%~50% patient is postoperative to be occurred again
Hair or transfer.
The monitoring of Diagnosis of Bladder at present, especially postoperative recurrence, mainly with urine sediment inspection and Urinary Bladder
Based on spectroscopy.However, cystoscopy have it is invasive, inconvenient, patient is more painful, also infection, bleeding equivalent risk,
And testing cost is expensive.Urine sediment checks that specificity is good but sensibility is low, is easy by factors such as urinary tract infections
Interference.Therefore both inspection methods are all subject to certain restrictions in clinical application.
In recent years, domestic and foreign scholars start to be dedicated to find sensitive, special, reliable, effective carcinoma of urinary bladder life from urine
Substance markers object simultaneously establishes noninvasive testing and inventive method to improve carcinoma of urinary bladder early diagnostic rate.
In the past to the detection of tumor markers in urine, the side such as PCR, ELISA, immunochromatography fluorescent marker is generally used
Method.PCR and ELISA method need to pre-process urine, to extract nucleic acid and protein, not being suitable for quickly detecting, and layer is immunized
Although analysis method is quick, the ratio of false negative is big, can only qualitative or sxemiquantitative, specificity and sensitivity have to be hoisted.Cause
This, needs the means for combining new method and technology new material exploitation novel bladder cancer urine early detection, raising to examine carcinoma of urinary bladder
Disconnected specificity and sensibility.
Invention content
Nuclear matrix protein NMP22 (NuMA) is to form the holder of nucleus internal structure, and same DNA replication dna, RNA are closed
It is closely related at the connection of, hormone, gene expression regulation.Massive tumor Apoptosis and NMP22 is released into urine when carcinoma of urinary bladder, made
Its level can increase 25 times.When with 10kU/mL being critical, it is 69.7% to the susceptibility of Diagnosis of Bladder, and specificity is
78.5%, the susceptibility to invasive bladder cancer diagnosis is 100%.Using the horizontal 10U/mL of urine NMP22 as critical value, wing is diagnosed
The sensibility of Guang transitional cell carcinoma is 88.3%, and specificity is 84.0%.Using 6.4U/mL as critical value, bladder transitional cell carcinoma is diagnosed
Sensibility be 84.6%, specificity be 74.2%, with urinary cytology check joint-detection sensibility can increase to 91%.Urine
NMP22 has higher specificity as the tumor marker of bladder transitional cell carcinoma, and can react the evil of tumour to a certain extent
Property degree and prognosis.
Bladder tumor antigen (bladder tumor anti-gen, BTA) is the proteolytic enzyme secreted by tumor of bladder,
It is IV Collagen Type VIs, fibronectin and laminin by bladder substrate membrane degradation, substrate is formed by with urine discharge
Membrane complex.The ingredient of this kind of catabolite is mainly molecular weight (16~165) × 103Specific polypeptides.Through cystoscope
In the bladder cancer patients made a definite diagnosis with pathological biopsy, the susceptibility of BTA is 67%, specificity 78%, and accuracy 73% is positive
Predicted value is 70%, negative predictive value 75%, and susceptibility has the raising by stages with carcinoma of urinary bladder and raised trend [Xie Ke
Base, bladder tumor antigen detect the clinical value of carcinoma of urinary bladder].It can be used as one of the qualitative index of transitional cell bladder carcinoma detection.
CK20 Cytokeratins 20 (Cytokeratin20, CK20) are one kind of I type keratoprotein intermediate filaments, are being tieed up
It holds the integrality of cell, the utilization for adjusting plasmosin, cellular signal transduction, cell differentiation, induction chemotherapy of tumors drug resistance, participate in
It plays an important role in the generation of tumour, development and cell surface receptor.CK20 skin and the uropoiesis of tumor of bladder on the gastrointestinal tract
Road epithelium has expression, and the expression of normal structure epithelium is severely limited.Have studies have shown that CK20 monoclonal antibodies
It connect, the CK20 monoclonal antibodies of colloid gold label are made and are adsorbed on nitrocellulose membrane with colloidal gold, it will using double faced adhesive tape
The nitrocellulose membrane is fixed on plastics negative and test strip is made.Simultaneously urine mark is detected with the test strips, UBC, BTA
This, tumor group is 51 BC patient's Urine specimens, and the urologic disease blood urine of 19 non-tumours is benign disease control group, and 20 strong
Health human urine is Normal group, compares three's testing result to evaluate CK20 monoclonal antibody colloidal gold strip clinical applications
Value.Result of study confirms that CK20 monoclonal antibody test strips are easy with detection method, quick, and stability, accuracy, specificity
The advantages that good and high sensitivity, non-invasive, there is preferable clinical value, is convenient for clinical expansion, has a wide range of applications
Foreground.
TERT (telomerase reverse transcriptase, reverse transcriptase of telomere) refers to having in Telomerase
The catalytic subunit of reverse transcriptase activity belongs to a part for Telomerase.And Telomerase refers to being had by what protein and RNA were formed
It is catalyzed the ribonucleoprotein particle that telomere synthesis extends.Telomerase synthesizes the DNA fragmentation TTAGGG of telomere repeatedly, can make telomere
It is not lost because of cell division, to make fissional number increase, (normal cell division number is limited, and tumour is thin
Born of the same parents have immortalization trend).Generally acknowledged, the telomerase activation of kinds of tumor cells is higher.And gene sequencing find, carcinoma of urinary bladder and
There are the TERT gene promoters in the mutation of moiety site, especially carcinoma of urinary bladder for TERT gene promoter regions in other tumours
The change that the frequency of mutation of son is up to 55.6%, TERT gene promoters makes the expression quantity of TERT increase, to make telomere enzyme activity
Property enhancing, and then induce and promote the canceration of the tumours such as carcinoma of urinary bladder.The detection of TERT promoter mutations generally by sequencing,
PCR is realized, can also be attempted by detecting the expression quantity of the expression quantity of the mRNA of TERT genetic transcriptions or Telomerase in urine
To carry out the early screening of tumour.
Rationally joint can play supplement using bladder cancer biomarkers object in above-mentioned four kinds of urines as Non-invasive detection means
The effect of cystoscope can be by checking urine tumour when lesion is small or can not be seen by cystoscope due to position relationship
Marker finds carcinoma of urinary bladder, or sensibility adjustment check cystoscope by urine tumor marker interval time.
With the development of quantum techniques, quantum dot (Quantum dots, QDs) is as a kind of novel semiconductor nano material
Material, it has also become the powerful of Molecular Detection and medical diagnosis, application range include cell, tissue mark's imaging, Cellular tracking,
Living imaging, medical diagnosis on disease and food safety detection etc..Quantum dot has the characteristics that：(1) luminescent color is different, can use single
One light source realizes polychrome detection；(2) while a variety of quantum dots of excitation are not easy to be overlapped, and the while of realizing multiple molecular is facilitated to examine
It surveys；(3) quantum yield is high, luminous intensity is strong, and photochemical stability is good, acceptable to excite repeatedly for a long time.Utilize quantum dot pair
Microballoon carries out encoding latter made protein chip, and the fluorescence power after being excited according to quantum dot carries out quantitative analysis, real
It is detected while existing multiple carcinoma of urinary bladder molecular targets.
It is an object of the invention to disclose protein chip that is a kind of while detecting four bladder carcinoma markers in urine.
The purpose of the present invention is what is be achieved through the following technical solutions：
Protein chip that is a kind of while detecting four bladder carcinoma markers in urine, wherein the protein chip is simultaneously
Protein chip containing NMP22 microballoons, BTA microballoons, CD20 microballoons and TERT microballoons；
The NMP22 monoclonals that the NMP22 microballoons are coated with microballoon and are coupled by the colloidal gold for being embedded in quantum dot resist
Body forms；
The BTA monoclonal antibody groups that the BTA microballoons are coated with microballoon and are coupled by the colloidal gold for being embedded in quantum dot
At；
The CD20 monoclonal antibodies that the CD20 microballoons are coated with microballoon and are coupled by the colloidal gold for being embedded in quantum dot
Composition；
The TERT monoclonal antibodies that the TERT microballoons are coated with microballoon and are coupled by the colloidal gold for being embedded in quantum dot
Composition.
Protein chip that is a kind of while detecting four bladder carcinoma markers in urine, the protein chip pass through following methods
It prepares：
(1), the preparation of colloidal gold coating antigen microballoon and the insertion of quantum dot；The quantum dot has same excitation wavelength
But launch wavelength is different；
(2), monoclonal antibody is prepared using the recombinant protein of NMP22, BTA, CD20 and TERT respectively as antigen；Dan Ke
The coupling with the coated former microballoon of the colloidal gold of embedded quantum dot respectively of grand antibody, it is molten to obtain NMP22 microspheres solutions, BTA microballoons
Liquid, CD20 microspheres solutions and TERT microspheres solutions；
(3), NMP22 microspheres solutions, BTA microspheres solutions, CD20 microspheres solutions and TERT microspheres solutions are taken respectively, in electricity
The protein chip of four bladder carcinoma markers in urine is mixed well to get while detected under the lasting stirring of magnetic stirring instrument.
Protein chip that is a kind of while detecting four bladder carcinoma markers in urine described in above-mentioned technical proposal, wherein
The detailed process of the preparation of the step (1) colloidal gold coating antigen microballoon and the insertion of quantum dot is：
(1), the preparation of colloidal gold；
(2), colloidal gold coating antigen microballoon：Colloidal gold and the 0.05g that step (1) preparation gained is added in centrifuge tube are micro-
Ball centrifuges after fully vibrating mixing, discards supernatant liquid, after precipitation is washed with distilled water and absolute ethyl alcohol repeatedly, at room temperature
The coated former microballoon of colloidal gold is obtained after drying；
(3), quantum dot is embedded in colloidal gold coating antigen microballoon：Teat glass is taken, 2mL quantum dot chloroformic solutions are separately added into
The coated former microballoon of colloidal gold that gained is prepared with 0.01g steps (2), fully there's almost no free amount in oscillation to solution
It is sub-, in draught cupboard after chloroform volatilization completely, remaining microballoon is washed repeatedly with absolute ethyl alcohol, after being dried at room temperature for
It obtains and is coated with microballoon in the colloidal gold for being embedded in quantum dot.
Protein chip that is a kind of while detecting four bladder carcinoma markers in urine described in above-mentioned technical proposal, wherein
Colloidal gold prepare detailed process be：Gold chloride is configured to 0.01% aqueous solution, is taken in 100mL to 250mL beakers, is heated
To boiling, keeps that sodium citrate is added in the state of boiling, stop after the red that the color of aqueous solution of chloraurate is stable
Heating, after red aqueous solution is cooled to room temperature, with distilled water restore to original volume to get；
Protein chip that is a kind of while detecting four bladder carcinoma markers in urine described in above-mentioned technical proposal, wherein
The monoclonal antibody detailed process with the coupling of the coated former microballoon of the colloidal gold of embedded quantum dot respectively in the step (2)
For：
(a), to NMP22, BTA, CD20 and TERT monoclonal antibody being dissolved in phosphate buffer, following places are carried out respectively
Reason：The solution of a concentration of 30ug/mL is diluted under the lasting stirring of electromagnetic agitation instrument；
(b), 1% Tween 80, the middle insertion quantum for preparing gained of 0.01g steps (3) are sequentially added into step (a)
The coated former microballoon of colloidal gold of point after persistently stirring 15min, is added and has not been coupled on 5% BSA solution closing microballoon
Site after the completion of to be closed, centrifuges solution, after taking precipitation PBS buffer solution to wash repeatedly, respectively obtains coupling
The NMP22 microballoons of NMP22 monoclonal antibodies, the BTA microballoons for being coupled BTA monoclonal antibodies, have been coupled CD40 monoclonal antibodies
CD20 microballoons and be coupled the TERT microballoons of TERT monoclonal antibodies.
The invention has the advantages that：
The protein chip of four bladder carcinoma markers in urine is detected while proposed by the invention, it is high for specificity
NMP22, BTA, CD20 and TERT, the early detection and recurrence for carrying out carcinoma of urinary bladder detect.The chip fabrication process is relatively easy, can
Mass production, cheap, chip detection means is quick, time saving and energy saving, is suitble to wide clinical application.
Description of the drawings：
1, Fig. 1 is the preparation method flow diagram of protein chip of the present invention.
2, Fig. 2 is the knot of the coated microballoon of colloidal gold through embedded quantum dot of the present invention for being coupled monoclonal antibody
Structure schematic diagram.
3, Fig. 3 is the structural schematic diagram of protein chip of the present invention.
Specific implementation mode：
To make technical scheme of the present invention be easy to understand, urine is detected simultaneously to one kind of the invention below in conjunction with specific test example
The protein chip of four bladder carcinoma markers is further described in liquid.
Protein chip of the present invention, by being coupled, there are four the quantum dot colloidal gold of marker antibody to be checked is coated micro-
Ball is constituted.In the present invention, the microballoon of not coated processing is named as former microballoon, it, respectively will packet according to the difference of marker
Microballoon name NMP22 microballoons, BTA microballoons, CD20 microballoons and TERT microballoons by after.The kernel of former microballoon is that diameter is identical poly-
Styrene, and so that it is taken elecrtonegativity by grafting processing.Former microsphere surface is coated with the colloidal gold embedded with quantum dot, goes forward side by side
One step is coupled the monoclonal antibody of marker to be measured by covalent mode respectively, and it is micro- to form NMP22 microballoons, BTA microballoons, CD20
Ball and TERT microballoons.
The NMP22 microballoons, BTA microballoons, CD20 microballoons and TERT microsphere surfaces are coated with the colloid embedded with quantum dot
The excitation wavelength of gold, embedded quantum dot is the same but launch wavelength is different.NMP22 microballoons are coupled by covalent manner
NMP22 monoclonal antibodies form NMP22 quantum dot fluorescence microballoons, and BTA microballoons are coupled BTA monoclonal antibody shapes by covalent manner
At BTA quantum dot fluorescence microballoons, CD20 microballoons are coupled CD20 monoclonal antibodies by covalent manner and form CD20 quantum dot fluorescences
Microballoon, TERT microballoons are coupled TERT monoclonal antibodies by covalent manner and form TERT quantum dot fluorescence microballoons.
Specifically, the preparation and detection of the protein chip are accomplished by the following way.
Embodiment 1：Colloidal gold is coated with the preparation of microballoon and the insertion of quantum dot：
1, the preparation of colloidal gold
The colloid gold particle prepared by the following method in the present invention, grain size are about 20nm, wherein the concentration of gold is about
0.1mg/mL。
Specifically preparation method is：First by gold chloride (HAUC14) it is configured to 0.01% aqueous solution, take 100mL to 250mL to burn
In cup, beaker washes down simultaneously silicidation using preceding through pickling, distilled water.After being heated to boiling, keep being added in the state of boiling
Sodium citrate and the color change for observing aqueous solution of chloraurate, it is seen that aqueous solution of chloraurate becomes grey from faint yellow, then becomes
It for black, then is gradually transitions red and keeps stablizing constant, the entire process have about 3min.After red aqueous solution is cooled to room temperature, use
Distilled water restore to original volume to get.
2, the coating of former microballoon：
Microsphere surface in the present invention carries elecrtonegativity, can attract the colloidal gold of above-mentioned preparation in its table by electrostatic interaction
Face deposits, to realize the coating of microballoon.
Specific implementation method is：0.05g microballoons and suitable colloidal gold are added in 15mL centrifuge tubes, fully vibrates mixing
After centrifuge, discard supernatant liquid, after precipitation is washed with distilled water and absolute ethyl alcohol repeatedly, colloidal gold obtained after being dried at room temperature for
Coated original microballoon, it is spare.
3, the insertion of quantum dot：
Same excitation wavelength but the mutually different four parts of quanta point materials of launch wavelength are chosen, is stored in a free form
In chloroformic solution.Clean 5mL teat glass is taken, 2mL quantum dots chloroformic solution is separately added into and about 0.01g colloidal golds is coated
Former microballoon fully there's almost no free quantum dot in oscillation to solution, in draught cupboard after chloroform volatilization completely, will remain
Under microballoon washed repeatedly with absolute ethyl alcohol, after being dried at room temperature for obtain be embedded in quantum dot colloidal gold be coated with microballoon,
It is spare.
Embodiment 2：The preparation of monoclonal antibody and coupling with the coated former microballoon of the colloidal gold of embedded quantum dot：
1, the preparation of cell fusion and hybridoma
6-8 week old female Balb/c mouse are chosen, the recombinant protein of NMP22, BTA, CD20 and TERT are injected intraperitoneally respectively
As antigen, make that antigen passes through blood circulation or Lymphatic Circulation enters peripheral immune organ, corresponding bone-marrow-derived lymphocyte stimulated to clone,
Make its activation, proliferation, and breaks up as sensitization bone-marrow-derived lymphocyte.After three days, using CO2Gas puts to death mouse, under sterile working
Spleen is taken out, splenocyte suspension is prepared into.The murine myeloma cell of logarithmic growth phase presses 5 with splenocyte：1 ratio,
It is mixed in the sterile nontoxic environment of 40 DEG C of water-baths, 50% polyethylene glycol (PEG) is added as fusion agent is promoted, makes lymphocyte and bone
Myeloma cells occur fusion and form hybridoma, and hybridoma is placed on HAT culture mediums and is cultivated, is changed the liquid once within every 3 days, observe
Hybridoma generation situation.
2, the screening and cloning of positive hybridoma：
After hybridoma appearance, Aspirate supernatant checks antibody.It is determined as the positive hybridoma of energy specific secretion antibody
Afterwards, it takes the hybridoma of continued growth to carry out proliferation passage, in succeeding generations, HAT is replaced with 10%FCS, passage cell is protected
It is stored in liquid nitrogen and carries out cloning.It is required for checking antibody per generation hybridoma, avoids the variation of positive cell and lose
It loses.
3, the preparation of monoclonal antibody ascites and the purifying of antibody
After taking Balb/c mouse, intraperitoneal injection 0.5mL atoleines to carry out pretreatment 1-2 weeks, respectively in intraperitoneal inoculation
Positive hybridoma cell is observed 1-2 weeks, after mouse web portion obviously expands, is extracted ascites with syringe, is collected repeatedly for several times,
Obtain the ascites containing a large amount of monoclonal antibodies.Ascites is all made of octanoic acid-ammonium sulfate precipitation method and is purified, purified precipitation
Monoclonal antibody is dissolved in the phosphate buffer solution of 10umol/L.Each monoclonal is identified by polyacrylamide gel electrophoresis
The purity of antibody, and the potency of each monoclonal antibody is measured using brief introduction ELISA method.According to the method for the invention system
Standby and purifying antibody titer 1：521000-1：124000.It is preserved by purity and the monoclonal antibody of titration
It is spare in -80 DEG C of refrigerators.
4, the coupling of monoclonal antibody and the coated former microballoon of colloidal gold of embedded quantum dot
The monoclonal antibody that will be dissolved in phosphate buffer respectively is diluted to concentration under the lasting stirring of electromagnetic agitation instrument
For the solution of 30ug/mL, 1% Tween 80 is sequentially added, 0.01g has been inserted into the coated former microballoon of colloidal gold of quantum dot,
After persistently stirring 15min, the site not being coupled on 5% BSA solution closing microballoon is added, after the completion of to be closed, to molten
Liquid is centrifuged, after taking precipitation PBS buffer solution to wash repeatedly, respectively obtain be coupled NMP22 monoclonal antibodies NMP22 it is micro-
Ball, the BTA microballoons for being coupled BTA monoclonal antibodies have been coupled the CD40 microballoons of CD40 monoclonal antibodies and to be coupled TERT mono-
The TERT microballoons of clonal antibody, each microballoon are stored in PBS buffer solution, and microballoon concentration is held in 1ug/mL or so.
5, the preparation of protein chip：
Equivalent NMP22 microspheres solutions, BTA microspheres solutions, CD20 microspheres solutions and TERT microspheres solutions are taken respectively, in electromagnetism
It stirs and is mixed well under the lasting stirring of instrument to get being preserved in -80 DEG C of refrigerators, in case detecting in urine sample four simultaneously
Bladder carcinoma marker (NMP22, BTA, CD20 and TERT) is used.
Embodiment 3：The detection of four bladder carcinoma markers of protein chip pair prepared by the present invention：
1, the acquisition of sample to be tested：
The detection of the micro marker suitable for urine of protein chip prepared by the present invention.Midstream urine from collection morning, immediately
Laboratory is transferred to, Cord blood is spare, in completion detection in 12h.
2, the total incubation of protein chip and sample to be tested：
The protein chip gone bail in there are PBS buffer solution makes to form microsphere suspensions after concussion fully.One in culture dish
The secondary protein chip that urine specimen and suspension state is added, after mixing well, in being protected from light 45min in 37 DEG C of incubators.It takes out
Appropriate microsphere suspensions are added after culture dish again, after mixing well, in being protected from light 30min in 37 DEG C of incubators.
3, the detection of four bladder carcinoma markers：
The total sample for being incubated and completing is taken, is placed on multi-functional stream type cell analyzer and detects, it is quasi- to carry software with analyzer
Dose-response curve is closed, the concentration of four bladder carcinoma markers in urine specimen to be measured is calculated.It repeats detection three times, takes each mark
The average of will object concentration is as reported concentrations.
The above, only presently preferred embodiments of the present invention, not to the present invention make in any form with substantial limit
System, all those skilled in the art, without departing from the scope of the present invention, when using disclosed above skill
Art content, and the equivalent variations for a little variation, modification and evolution made are the equivalent embodiment of the present invention；Meanwhile it is all according to
According to the substantial technological of the present invention to the variation, modification and evolution of any equivalent variations made by above example, this is still fallen within
In the range of the technical solution of invention.
Claims (5)
1. protein chip that is a kind of while detecting four bladder carcinoma markers in urine, it is characterised in that：The protein chip is
Protein chip containing NMP22 microballoons, BTA microballoons, CD20 microballoons and TERT microballoons simultaneously；
The NMP22 monoclonal antibody groups that the NMP22 microballoons are coated with microballoon and are coupled by the colloidal gold for being embedded in quantum dot
At；
The BTA monoclonal antibodies that the BTA microballoons are coated with microballoon and are coupled by the colloidal gold for being embedded in quantum dot form；
The CD20 monoclonal antibodies that the CD20 microballoons are coated with microballoon and are coupled by the colloidal gold for being embedded in quantum dot form；
The TERT monoclonal antibodies that the TERT microballoons are coated with microballoon and are coupled by the colloidal gold for being embedded in quantum dot form.
2. protein chip that is a kind of while detecting four bladder carcinoma markers in urine, the protein chip pass through following methods system
It is standby：
(1), the preparation of colloidal gold coating antigen microballoon and the insertion of quantum dot；The quantum dot has same excitation wavelength but hair
Ejected wave length is different；
(2), monoclonal antibody is prepared using the recombinant protein of NMP22, BTA, CD20 and TERT respectively as antigen；Monoclonal is anti-
The body coupling with the coated former microballoon of the colloidal gold of embedded quantum dot respectively, obtain NMP22 microspheres solutions, BTA microspheres solutions,
CD20 microspheres solutions and TERT microspheres solutions；
(3), NMP22 microspheres solutions, BTA microspheres solutions, CD20 microspheres solutions and TERT microspheres solutions are taken respectively, are stirred in electromagnetism
Mix the protein chip for mixing well to get while detecting four bladder carcinoma markers in urine under the lasting stirring of instrument.
3. protein chip that is according to claim 2 a kind of while detecting four bladder carcinoma markers in urine, feature
It is, the detailed process of the preparation of the step (1) colloidal gold coating antigen microballoon and the insertion of quantum dot is：
(1), the preparation of colloidal gold；
(2), colloidal gold coating antigen microballoon：Colloidal gold and 0.05g microballoons that step (1) prepares gained are added in centrifuge tube, fills
It is centrifuged after dividing oscillation mixing, supernatant liquid is discarded, after precipitation is washed with distilled water and absolute ethyl alcohol repeatedly, after being dried at room temperature for
Obtain the coated former microballoon of colloidal gold；
(3), quantum dot is embedded in colloidal gold coating antigen microballoon：Take teat glass, be separately added into 2mL quantum dots chloroformic solution and
0.01g steps (2) prepare the coated former microballoon of colloidal gold of gained, fully there's almost no free quantum in oscillation to solution
Remaining microballoon is washed with absolute ethyl alcohol, is obtained after being dried at room temperature in draught cupboard after chloroform volatilization completely by point repeatedly
It obtains and is coated with microballoon in the colloidal gold for being embedded in quantum dot.
4. protein chip that is according to claim 2 or 3 a kind of while detecting four bladder carcinoma markers in urine, special
Sign is that detailed process prepared by colloidal gold is：Gold chloride is configured to 0.01% aqueous solution, takes 100mL to 250mL beakers
In, it after being heated to boiling, keeps that sodium citrate is added in the state of boiling, when the color of aqueous solution of chloraurate is stable red
Afterwards stop heating, after red aqueous solution is cooled to room temperature, with distilled water restore to original volume to get.
5. protein chip that is according to claim 2 a kind of while detecting four bladder carcinoma markers in urine, feature
It is, monoclonal antibody is specific with the coupling of the coated former microballoon of the colloidal gold of embedded quantum dot respectively in the step (2)
Process is：
(a), to NMP22, BTA, CD20 and TERT monoclonal antibody being dissolved in phosphate buffer, following processing are carried out respectively：
The solution of a concentration of 30ug/mL is diluted under the lasting stirring of electromagnetic agitation instrument；
(b), 1% Tween 80 is sequentially added into step (a), the insertion quantum dot of gained is prepared in 0.01g steps (3)
After persistently stirring 15min, the position not being coupled on 5% BSA solution closing microballoon is added in the coated former microballoon of colloidal gold
Point after the completion of to be closed, centrifuges solution, after taking precipitation PBS buffer solution to wash repeatedly, respectively obtains coupling
The NMP22 microballoons of NMP22 monoclonal antibodies, the BTA microballoons for being coupled BTA monoclonal antibodies, have been coupled CD40 monoclonal antibodies
CD20 microballoons and be coupled the TERT microballoons of TERT monoclonal antibodies.
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| CN201711007608.7A CN108414755A (en) | 2017-10-25 | 2017-10-25 | Protein chip that is a kind of while detecting four bladder carcinoma markers in urine |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111190013A (en) * | 2020-02-27 | 2020-05-22 | 郑州大学第一附属医院 | Group of esophageal cancer detection markers and application thereof in preparation of esophageal cancer screening kit |
| CN112924683A (en) * | 2019-12-05 | 2021-06-08 | 张曼 | Application of urine exosome CD40 protein and polypeptide fragment thereof in bladder cancer |
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| CN101718792A (en) * | 2009-12-10 | 2010-06-02 | 兰州大学 | Protein chip for diagnosing bladder tumors |
| CN101730848A (en) * | 2007-03-20 | 2010-06-09 | 因达斯生物有限公司 | Method for the diagnosis and/or prognosis of cancer of the bladder |
| CN104865383A (en) * | 2015-01-12 | 2015-08-26 | 深圳市森塔医疗器械有限公司 | Liquid phase protein chip for combined detection of five colorectal cancer markers, and preparation method thereof |
| CN105891500A (en) * | 2015-01-16 | 2016-08-24 | 刘晓强 | Test paper for fast detecting carcinoma of urinary bladder |
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| CN101730848A (en) * | 2007-03-20 | 2010-06-09 | 因达斯生物有限公司 | Method for the diagnosis and/or prognosis of cancer of the bladder |
| CN101718792A (en) * | 2009-12-10 | 2010-06-02 | 兰州大学 | Protein chip for diagnosing bladder tumors |
| CN104865383A (en) * | 2015-01-12 | 2015-08-26 | 深圳市森塔医疗器械有限公司 | Liquid phase protein chip for combined detection of five colorectal cancer markers, and preparation method thereof |
| CN105891500A (en) * | 2015-01-16 | 2016-08-24 | 刘晓强 | Test paper for fast detecting carcinoma of urinary bladder |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112924683A (en) * | 2019-12-05 | 2021-06-08 | 张曼 | Application of urine exosome CD40 protein and polypeptide fragment thereof in bladder cancer |
| CN112924683B (en) * | 2019-12-05 | 2023-08-29 | 张曼 | Application of urine exosome CD40 protein and polypeptide fragment thereof in bladder cancer |
| CN111190013A (en) * | 2020-02-27 | 2020-05-22 | 郑州大学第一附属医院 | Group of esophageal cancer detection markers and application thereof in preparation of esophageal cancer screening kit |
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