CN104316683B - For the ovarian cancer cell detection kit of whole blood - Google Patents
For the ovarian cancer cell detection kit of whole blood Download PDFInfo
- Publication number
- CN104316683B CN104316683B CN201410538494.9A CN201410538494A CN104316683B CN 104316683 B CN104316683 B CN 104316683B CN 201410538494 A CN201410538494 A CN 201410538494A CN 104316683 B CN104316683 B CN 104316683B
- Authority
- CN
- China
- Prior art keywords
- qds
- bsa
- add
- ovarian cancer
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57449—Specifically defined cancers of ovaries
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides the ovarian cancer cell detection kit for whole blood. Described kit mainly comprises HE4 goat polyclonal antibody and biotinylated bovine serum albumin (BSA)-folic acid (FA) that can specific recognition ovarian cancer cell people epididymal proteins (HE4) antigen, the nanometer magnetic bead (SA-IO) of streptavidin, the QDs(SA-QDs of streptavidin), the anti-sheep IgG of the anti-HE4 rabbit of biotinylation and connect reagent etc. What described kit can utilize immunomagnetic beads can efficiently separate the cancer cell in ovarian cancer patients whole blood by enriched character, and by SA-QDs and biotinylated HE4 antigen antibody complex specific binding target ovarian cancer cell, realizes that multiple signal is collaborative to be amplified. This reagent has stronger sensitivity and specificity, has extremely wide application prospect in the earlier detection of oophoroma.
Description
Technical field
The invention belongs to biotechnology and biomedical sector. Particularly, the invention provides a kind of oophoroma of detectingKit.
Background technology
Oophoroma is gynaecology's common cancer, and its incidence of disease is arranged in the 3rd, female reproductive system tumour, case fatality rateBut occupy the first. When most of oophoromas are made a definite diagnosis, be late period, its 5 years survival rates are only 30%, early diagnosis and therapy ovaryCancer is to improve patient's prognosis, improves the key factor of its survival rate. At present, the common screening technique of oophoroma has 2 kinds clinically:1) Transvaginal Ultrasound inspection; 2) change of serum C A125 detects. All there is its limitation in the two. Research shows, in blood of cancer patientsThe appearance of circulating tumor cell (CTCs), early than visible solid tumor, therefore, is found in a kind of high specific enrichment human peripheral bloodThe method of CTCs is the focus of studying at present.
Immunity magnetic separation technique is one of important component part of CTCs fast separating concentration technology, and what this technology related to exempts fromEpidemic disease magnetic bead not only can binding active proteins matter or part but also can, by attraction, process antibody or part and magnetic through someAfter pearl combination, can be combined with target antigen specifically and form magnetic bead-antibody (part)-antigenic compound, then at externally-applied magnetic fieldEffect under by the object cell that contains target antigen and other cell separation, thereby reach the effect of separation and purification object cell.This technology has been widely used in diagnosis, treatment and the prognostic study of various tumours, but freshly in oophoroma earlier detection grinds lessStudy carefully. Because folacin receptor (FR) is extremely low in most of normal tissue expression levels, and at ovarian cancer cell surface expression height beThe binding affinity of 95%, FA and FR is 0.19nM, and therefore, FA is connected to Fe by the present invention2O3And Fe3O4Composite Nano crystalSurface, makes magnetic bead have target identification and Magnetic Isolation dual-use function, thereby realizes the enrichment of ovarian cancer cell, effectively improves inspectionSurvey sensitivity and realize earlier detection oophoroma.
People's epididymal proteins 4(HE4) be a kind of whey acidic 4-curing center (WFDC) albumen, this albumen is probably 20 ~25Kd. HE4 expression in ovarian cancer cell matter is high is 93%. An evaluation to the relevant large number of biological mark of oophoromaResearch shows that HE4 detects in early days and has the highest sensitivity in diagnosis of ovarian cancer, and its susceptibility is 82.7% and CA125Only have 45.9%. Therefore, HE4, as a kind of new tumor markers, can help to carry out earlier detection and the risk of oophoromaAssessment.
Quantum dot (QDs) is nanocrystalline as a kind of novel fluorescence, has some unique fluorescence properties, as quantum yieldHigh, photochemical stability is high good, is difficult for photodissociation etc. At present QDs is combined with antibody to prepare the report of fluorescence immunoassay probe a lot,But QDs immune fluorescent probe still exists in the time that very micro-material is detected that signal is low, detection sensitivity is low etc. asksTopic. Biotin-avidin system is a kind of novel signal magnifying tags technology, utilizes Avidin to modify after QDs, and QDs can be veryBe easy to be combined with biotinylated antibody, effectively amplify its fluorescence signal, detect thereby realize ultrasensitiveness ovarian cancer cell.
Summary of the invention
Key technical problem to be solved by this invention is to examine fast for ovarian cancer cell in the whole blood there is no at presentTest agent box provides a kind of kit based on Magnetic Isolation and QDs immunofluorescence label detection ovarian cancer cell. Utilize immunityMagnetic bead can enriched character and QDs fluorescence signal intensity change, set up that multiple signal is collaborative to be amplified, have supersensitive completeThe kit of fast detecting ovarian cancer cell in blood. This kit can improve resolution ratio, sensitivity and the spy that oophoroma detectsThe opposite sex, more effectively carries out earlier detection and the risk assessment of oophoroma.
Ovarian cancer cell detection kit for whole blood disclosed by the invention comprises: HE4 goat polyclonal antibody, biologyBovine serum albumin (the BSA)-folic acid (FA) of elementization, the nanometer magnetic bead (SA-IO) of streptavidin, streptavidinQDs(SA-QDs), the anti-sheep of biotinylation rabbit (HE4) IgG.
Described nanometer magnetic bead is Fe2O3And Fe3O4Compound nanometer magnetic bead, particle diameter is 25nm, surface carboxyl functionalized.HE4 goat polyclonal antibody and biotinylation BSA-FA respectively can specific recognition ovarian cancer cell matter in HE4 antigen and cellThe FR on film surface, wherein HE4 was the antigen that is expressed in (93%) on ovarian cancer cell matter golgiosome, it is in normal structureExpression is lower. FR at the expression on ovarian cancer cell surface up to 95%, and extremely low in most of normal tissue expression amounts.
Described SA-QDs, its preparation method is as follows: 1) to be dissolved in 100 μ LpH be 5.5 to 100 μ L8 μ mol/LQDsBB, according to QD:EDC mol ratio 1:1000, QD:NHS mol ratio 1:2000 adds EDC/NHS, room temperature reaction 5 ~ 10min; 2)Adjust pH value of solution to 8 ~ 8.5, add SA1.056mg, continue to mix after 2h, add the PBS sealing 10 of 10 μ L containing 5%BSAMin cessation reaction; 3) SA-QDs compound 10000r/min, centrifugal 5min removes after precipitation, uses the BB that 2mLpH is 7In 100K super filter tube, wash, be finally resuspended in 0.1mLpH and be in 7 BB.
Described biotinylation BSA-FA, its preparation method is as follows: 1) FA-PEG-NH2Coupling: FA6mg is dissolved inIn 600 μ L dimethyl sulfoxide (DMSO)s, add N, N-dicyclohexylcarbodiimide (DCC) 3mg, N, N-hydroxy-succinamide (NHSS)3mg and the common darkroom reaction of triethylamine 60 μ L 4h, add the reaction of two amino polyethylene glycol 30mg darkroom to spend the night; React completelyCentrifugal afterwards, use-20 DEG C of acetone precipitations, ethyl acetate washing 3 times, is placed in vent cabinet and is dried and keeps in Dark Place; 2) FA-PEG-The coupling of BSA: FA-PEG-NH25mg, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) 2.5Mg, NHSS2.5mg are dissolved in common room temperature reaction 10min in the borate buffer (BB) that 500 μ LpH are 7; Add BSA8.35mg, room temperature reaction 2h, after dialysis purifying 3 times in 4 DEG C of Refrigerator stores; 3) preparation of biotinylation BSA-FA: press long-chainBiotin: incubated at room 30min after FA-PEG-BSA mol ratio 20:1 mixes, dialyses 3 by mixed solution in 4 DEG C of ultra-pure watersD, 4 DEG C save backup.
Described biotinylation BSA-FA utilizes the upper abundant amino active group of large molecule BSA, and volume is rawIt is upper that thing element and FA are enriched in BSA, is combined quantity realization amplification thereby increase FA and Cell binding probability and biotin with IO-SA.
The anti-sheep IgG of the anti-HE4 rabbit of described biotinylation, its preparation method is as follows: 1) get biotin 15mg, EDCIt is in 6.5 phosphate buffers (PBS) that 2.4mg, NHSS3.6mg are dissolved in 2mL0.02MpH; 2) add anti-HE4 rabbit anti-Sheep IgG5.3mg, room temperature is placed in and on blending instrument, stirs 30min; 3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving,The 1d that dialyses in PBS and deionized water ,-20 DEG C of storages.
Described SA-IO is prepared via covalent bond coupling by SA and IO; Its preparation method is as follows: 1) 400 μ L5mg/mLIO is dissolved in the BB that 400 μ LpH are 5.5, and according to IO:EDC mol ratio 1:1000, IO:NHS mol ratio 1:2000 adds EDC/NHS, room temperature reaction 5 ~ 10min; 2) adjust pH value of solution to 8 ~ 8.5, add SA0.08mg, continue to mix after 2h, add 20μ L is containing the PBS sealing 10min cessation reaction of 5%BSA; 3) adding appropriate pH is that 7 BB mixes, and separates in 1.0 teslas' magneticSeparation and purification IO-SA10 ~ 24h in device, repeats to be resuspended in after 2 times 1mLpH and is in 7 BB, and new system magnetic bead concentration is 2mg/ML. Wherein, 5%BSA/PBS can be combined by the carboxyl of IO or QDs remained on surface in coupled product of the active amino on BSA,Reduce the non-specific adsorption of coupled product.
Described kit also comprises fixer (4% paraformaldehyde/trihydroxy aminomethane-hydrochloride buffer (TBS) pH7.6), penetrating fluid (TBS+0.1% Tween-20 pH7.6), confining liquid (0.1% casein+5%BSA+TBSpH7.6), washingLiquid (TBSpH7.6).
Described detection sample is whole blood.
Described QDs is CdSSe/ZnS, and emission wavelength is 550nm.
The invention discloses the application of mentioned reagent box enrichment ovarian cancer cell in whole blood, and repair by specific antibodyThe QDs of decorationsization carries out fluorescence labeling, observes counting separative efficiency and fluorescence signal acquisition system fixed by inverted fluorescence microscopeComponent analysis obtains final detection result.
Beneficial effect: oophoroma case fatality rate in gynecologic malignant tumor is in first, sets up easy, oophoroma fastCell detection method is the early detection to tumour not only, and also the rapid evaluation to Chemotherapy of Tumor Patients medicine, individualized treatment (faceThe detection of bed sieve medicine, drug resistance), the monitoring of tumor recurrence and the exploitation of tumour new drug have great importance.
The present invention is antibody molecule and biotin coupling, avoided in conventional method, antibody molecule being coupled to QDs or magneticBead surface causes antibody activity to reduce and sterically hindered large shortcoming.
The present invention utilizes the upper abundant amino active group of large molecule BSA, and volume biotin and FA are enriched in to BSA above,Thereby increase FA and Cell binding probability and biotin and be combined quantity realization amplification with IO-SA.
The present invention is according to the double-antibody sandwich principle of fluorescence immunoassay, utilizes that immunomagnetic beads separating rate is fast, efficiency is high, operationThe advantage such as easy, high in conjunction with QDs photochemical stability, be difficult for the good optical characteristics such as photodissociation, respectively with immunomagnetic beads and QDsFor carrier connects biotinylated BSA-FA and the anti-sheep IgG of anti-HE4 rabbit, make the detection of cancer cell in whole blood quickly and accuratelyBe achieved.
Brief description of the drawings
The SKOV3 cell of Fig. 1 based on Magnetic Isolation and the separative efficiency figure of A549 cell, wherein SKOV3-2 is not for addingThe separative efficiency of SKOV3 cell when the simple IO-SA magnetic of biotinylation BSA-FA reaction separates.
The QDs immunofluorescence label coloration result of Fig. 2 SKOV3 cell and A549 cell.
Detailed description of the invention
Further illustrate the present invention by following examples, but claim of the present invention is not limited only to embodiment.
Following embodiment if no special instructions method therefor is conventional method.
The magnetic nano-particle SHP-05-25 using in embodiment and QD550 be purchased from OceanNanoTech company of the U.S.,Biotin, SA are purchased from Shanghai Chinese blue chemical company. HE4 goat polyclonal antibody (C-12) is purchased from Santa Cruz biotechnology public affairsDepartment, the anti-sheep of rabbit (HE4) IgG(BA1040) be purchased from doctor's moral biotech company, DCC, NHSS, NHS, EDC buys in the U.S.Sigma-Aldrich company, BSA is purchased from Biosharp company, folic acid purchased fromZhengzhou Song Hua commerce and trade Co., Ltd, be inverted fluorescence aobviousMicro mirror is Japanese Nikon company product, and the quantitative analysis of fluorescence signal acquisition system is the online ImageJ2x of download software analysis institute.
Nanometer magnetic bead is Fe2O3And Fe3O4Compound nanometer magnetic bead, particle diameter is 25nm, surface carboxyl functionalized.
Described QDs is CdSSe/ZnS, and emission wavelength is 550nm.
In embodiment, write a Chinese character in simplified form:
Quantum dot (QDs), ovarian cancer cell people epididymal proteins (HE4), bovine serum albumin (BSA), folic acid (FA), strepto-parentWith the nanometer magnetic bead (SA-IO) of element, the QDs(SA-QDs of streptavidin), folacin receptor (FR).
Fixer (4% paraformaldehyde/trihydroxy aminomethane-hydrochloride buffer (TBS) pH7.6), penetrating fluid (TBS+0.1% Tween-20 pH7.6), confining liquid (0.1% casein+5%BSA+TBSpH7.6), cleaning solution (TBSpH7.6).
Embodiment 1 detects oophoroma SKOV3 cell for the ovarian cancer cell detection kit of whole blood
1 prepares biotinylation BSA-FA
1）FA-PEG-NH2Coupling: FA6mg is dissolved in 600 μ L dimethyl sulfoxide (DMSO)s, adds DCC3mg, NHSS3mg and the common darkroom reaction of triethylamine 60 μ L 4h, add the reaction of two amino polyethylene glycol 30mg darkroom to spend the night; React completelyCentrifugal afterwards, use-20 DEG C of acetone precipitations, ethyl acetate washing 3 times, is placed in vent cabinet and is dried and keeps in Dark Place; 2) FA-PEG-The coupling of BSA: FA-PEG-NH25mg, EDC2.5mg, NHS2.5mg are dissolved in common room temperature in the BB that 500 μ LpH are 7Reaction 10min; Add BSA8.35mg, room temperature reaction 2h, after dialysis purifying 3 times in 4 DEG C of Refrigerator stores; 3) biotinylationThe preparation of BSA-FA: press long-chain biotin: incubated at room 30min after FA-PEG-BSA mol ratio 20:1 mixes, will mix moltenLiquid is in 4 DEG C of ultra-pure water dialysis 3d, and 4 DEG C save backup.
2 prepare the anti-sheep IgG of the anti-HE4 rabbit of biotinylation
1) getting biotin 15mg, EDC2.4mg, NHSS3.6mg, to be dissolved in 2mL0.02MpH be in 6.5PBS;2) add the anti-sheep IgG5.3mg of anti-HE4 rabbit, room temperature is placed in and on blending instrument, stirs 30min; 3) above-mentioned solution decompression is spin-dried for moltenAgent, deionized water dissolving, the 1d that dialyses in PBS and deionized water ,-20 DEG C of storages.
3 preparation SA-IO
1) 400 μ LIO(concentration are 5mg/mL) be dissolved in the BB that 400 μ LpH are 5.5, according to IO:EDC mol ratio 1:1000, IO:NHS mol ratio 1:2000 adds EDC/NHS, room temperature reaction 5 ~ 10min; 2) adjust pH value of solution to 8 ~ 8.5, addSA0.08mg, continues to mix after 2h, adds the PBS sealing 10min cessation reaction of 20 μ L containing 5%BSA; 3) add in right amountPH is that 7 BB mixes, and separation and purification IO-SA10 ~ 24h in 1.0 tesla's magnetic separators is resuspended in 1mL after repeating 2 timesPH is that in 7 BB, new system magnetic bead concentration is 2mg/mL.
4 preparation SA-QDs
1) 100 μ LQDs(concentration are 8 μ M) be dissolved in the BB that 100 μ LpH are 5.5, according to QD:EDC mol ratio 1:1000, QD:NHS mol ratio 1:2000 adds EDC/NHS, room temperature reaction 5 ~ 10min. 2) adjust pH value of solution to 8 ~ 8.5, addSA1.056mg, continues to mix after 2h, adds the PBS sealing 10min cessation reaction of 10 μ L containing 5%BSA. 3) QD-SA is multipleCompound 10000r/min, centrifugal 5min removes after precipitation, uses the BB that 2mLpH is 7 to wash in 100K super filter tube,After to be resuspended in 0.1mLpH be in 7 BB.
The IO(IO-FA of 5 preparation FA functionalized modifications)
1）FA-PEG-NH2Coupling: FA6mg is dissolved in 600 μ L dimethyl sulfoxide (DMSO)s, adds DCC3mg, NHSS3mg and the common darkroom reaction of triethylamine 60 μ L 4h, add the reaction of two amino polyethylene glycol 30mg darkroom to spend the night; 2) IO withFA-PEG-NH2Coupling: 200 μ LIO(concentration are 5mg/mL) be dissolved in the BB that 200 μ LpH are 5.5, according to IO:EDC moleThan 1:1000, IO:NHS mol ratio 1:2000 adds EDC/NHS, room temperature reaction 5 ~ 10min. 2) add 500 μ LpH be 8 ~8.5BB, adds FA-PEG-NH27mg, continues to mix after 2h, adds 10 μ L to stop containing the PBS sealing 10min of 5%BSAReaction; 3) adding appropriate pH is that 7 BB mixes, and separation and purification IO-FA10 ~ 24h in 1.0 tesla's magnetic separators, repeatsAfter 2 times, be resuspended in the BB that 500 μ LpH are 7, new system magnetic bead concentration is 2mg/mL.
The dyeing of 6SKOV3 cell and counting
1) obtain the oophoroma SKOV3 cell 4mL of exponential phase, add 8 μ LHoechest core dyestuffs, be placed in 37DEG C, hatch 30min in 5%CO2 incubator; 1000r/min, abandons supernatant after centrifugal 30s, adds PBS10mL to clean, and 1000r/min1min(repeated washing 2 times); Resuspended with 4mL3%BSA/PBS. 2) with cell counting count board counting (25 large lattice *16 little lattice) quantity of SKOV3 cell, under fluorescence microscope, count principle and be: under a line ball cell number left side is not several on right, the number of number.
7IO-FA magnetic nano-particle separation of whole blood SKOV3 cell
1) according to 1:109/ mL adds prestained SKOV3 cell in 1mL whole blood to, and on blending instrument, room temperature is fully mixedAfter even 1h, add IO-FA magnetic nano-particle according to IO-FA and FR mol ratio 20:1, continue to mix 30min, form compoundThing IO-FA-cell; 2) IO-FA-cell complexes is placed in to 1.0T magnetic separator magnetic and separates 1h, carefully abandon supernatant, with 50μ LPBS is resuspended to be placed in 24 porocyte culture plates.
8 magnetic nano-particle magnetic signals based on biotin-SA and BAS mediation amplify separation of whole blood SKOV3 cell
1) according to 1:109/ mL adds prestained SKOV3 cell in 1mL whole blood to, after fully mixing, according to lifeThing elementization BSA-FA and FR mol ratio are that 5:1 adds biotinylation BSA-FA, and on blending instrument, room temperature mixes 1h, formation compoundThing is biotinylation BSA-FA-cell; 2) add IO-SA according to IO-SA and biotinylation BSA-FA mol ratio 4:1, continue mixedEven 30min, the compound of formation is IO-SA-biotinylation BSA-FA-cell; 3) by thin IO-SA-biotinylation BSA-FA-Born of the same parents' compound be placed in 1.0T magnetic separator magnetic separate 1h, carefully abandon supernatant, with 50 μ LPBS resuspended be placed in 24 porocytes trainSupport in plate.
The quantum dot immune fluorescent dyeing of 9SKOV3 cell
1) adopt fixer fixed cell 15min at normal temperatures, use immediately cleaning solution rinsing 3 times; 2) use penetrating fluid normal temperatureLower permeation cell 20min, cleaning solution rinses the nonspecific binding site 1h using afterwards for 3 times on confining liquid closing cell; 3) removeConfining liquid, adds appropriate HE4 goat polyclonal antibody and cell to hatch altogether at normal temperatures 2h. Cleaning solution rinses 3 times (at every turn5min), add biotinylation-anti-HE4 rabbit anti-sheep IgG compound and cell to hatch altogether at normal temperatures 1h; 4) cleaning solution rinses 3Inferior (each 5min), adds appropriate SA-QDs compound reaction 30min, rinses and sloughs for 3 times after background colour in inversion fluorescence microscopyMicroscopic observation fluorescence signal is also carried out quantitative analysis and is calculated separative efficiency by fluorescence signal acquisition system.
The non-specific control experiment of 10 magnetic bead
1) select SKOV3 cell, step 1-4 is the same; 2) cell count is with step 6, the separation process of SKOV3 cellular whole blood magneticDo not add biotinylation BSA-FA, all the other operating procedures are with 8,9.
The non-specific control experiment of 11QDs
1) select SKOV3 cell, step 1-8 is the same. 2) quantum dot immune fluorescent of the SKOV3 cell after the separation of whole blood magneticIn dyeing course, do not add HE4 goat polyclonal antibody and biotinylation-anti-goat-anti nanocrystal composition of anti-HE4 rabbit, all the other steps are same9。
12 concurrent control group experiments
The lung cancer A549 cell of selecting FR to express feminine gender and the low expression of HE4, the same 1-9 of all the other operating procedures.
(experiment of above-mentioned steps 7,8,10,12 for carry out under equal conditions simultaneously, and experiment number is no less than 3 times; Step9 and 11 is the identification experiment simultaneously carrying out)
11 results:
1) Hoechst core dyestuff can fully be located oophoroma SKOV3 cell, via biotinylation-BSA-FA/IO-SA andThe SKOV3 of IO-FA immunity magnetic separation and the anti-sheep Antibody-antigen complex of QDs-biotinylation-anti-HE4 rabbit mark, cell numberAll more, wherein, biotinylation BSA-FA/IO-SA can be up to 87.1% through many experiments to the separative efficiency of SKOV3 cell, andThe separative efficiency of IO-FA is up to 43%, A549 cell capture number rareness under equivalent responses condition, and its capture rate only has23.3% and 23.1%. In addition, the non-specific experiment of magnetic bead shows, in the situation that not having FA to participate in, and the separative efficiency of IO-SAAll not high, its separative efficiency to SKOV3 cell is about 8.7% left and right. And to the separative efficiency of A549 cell also in 8.6% left and right(Fig. 1). Result shows, FA can specific recognition oophoroma SKOV3 cell, through the magnetic nano particle of biotin-SA and BAS mediationSub-magnetic signal amplification system can obviously improve cell capture efficiency, improves cell detection sensitivity. (Fig. 1)
2) via biotinylation-BSA-FA/IO-SA, and the immune magnetic of IO-FA separates and QDs-biotinylation-anti-HE4 rabbit resistsThe SKOV3 cell of sheep Antibody-antigen complex mark, equal visible significantly green fluorescence in cytoplasm, and magnetic separating trapA549 cell has no obvious green fluorescence after mark, does not add HE4 goat polyclonal antibody and biotinylation-anti-HE4 rabbit anti-Goat-anti nanocrystal composition, by the SKOV3 cell of the non-specific mark of SA-QDs, has no obvious green fluorescence in cytoplasm. (Fig. 2)
3), in clinical practice sample, 20 routine ovarian cancer patients peripheral bloods and 100 routine healthy women peripheral bloods use this reagentBox shows after detecting, finds circulating tumor cell, and do not send out in healthy women peripheral blood in 16 routine ovarian cancer patients peripheral bloodsExisting circulating tumor cell. (table 1)
Table 1
| Actual sample. | Pathological diagnosis | Blood volume/mL | Circulating tumor cell number/ |
| 1 | Mucous cystadenocarcinoma of ovary | 1 | 3 |
| 2 | Serous cystadenocarcinoma of ovary | 1 | 2 |
| 3 | Ovary Krukenberg knurl | 2 | 21 |
| 4 | Mucous cystadenocarcinoma of ovary | 1.2 | 1 |
| 5 | Serous cystadenocarcinoma of ovary | 1.5 | 8 |
| 6 | clear cell carcinoma | 1.5 | 0 |
| 7 | Serous cystadenocarcinoma of ovary | 1 | 5 |
| 8 | Serous cystadenocarcinoma of ovary | 1.2 | 16 |
| 9 | Boundary property serous cystadenocarcinoma | 1 | 1 |
| 10 | Mucous cystadenocarcinoma of ovary | 1 | 2 |
| 11 | Serous cystadenocarcinoma of ovary | 1 | 7 |
| 12 | Serous cystadenocarcinoma of ovary | 1 | 31 |
| 13 | Clear cell carcinoma | 1.5 | 0 |
| 14 | Boundary property MCAC | 1 | 0 |
| 15 | Boundary property serous cystadenocarcinoma | 1 | 1 |
| 16 | Serous cystadenocarcinoma of ovary | 1.5 | 4 |
| 17 | Dysgerminoma | 1 | 0 |
| 18 | Serous cystadenocarcinoma of ovary | 1 | 3 |
| 19 | Mucous cystadenocarcinoma of ovary | 2 | 1 |
| 20 | Serous cystadenocarcinoma of ovary | 1 | 16 |
| 21-120 | Healthy women | 1 | 0 |
The Magnetic Isolation and the QDs-SA/ biotin that the present invention is based on biotinylation BSA-FA/IO-SA are more than experimental results show thatThe immunofluorescence label of changing anti-HE4 antigen antibody complex can be used for specific enrichment oophoroma SKOV3 cell and it is carried outSpecificity checking, thus can be used for the detection of early ovarian cancer. After the inventive method detection oophoroma result is positive, Ke YijinThe employing other technologies means of one step are made a definite diagnosis oophoroma.
Embodiment 2 is the application in ovarian cancer patients whole blood for the ovarian cancer cell detection kit of whole blood
Get kit of the present invention: comprise 1) HE4 goat polyclonal antibody; 2) biotinylated BSA-FA; 3) chainThe nanometer magnetic bead SA-IO of mould Avidin; 4) QDs of streptavidin is SA-QDs; 5) the anti-sheep of the anti-HE4 rabbit of biotinylationIgG；
Wherein, each substances preparation method is as follows:
1 prepares biotinylation BSA-FA
1）FA-PEG-NH2Coupling: FA6mg is dissolved in 600 μ L dimethyl sulfoxide (DMSO)s, adds DCC3mg, NHS3Mg and the common darkroom reaction of triethylamine 60 μ L 4h, add the reaction of two amino polyethylene glycol 30mg darkroom to spend the night; After reacting completelyCentrifugal, use-20 DEG C of acetone precipitations, ethyl acetate washing 3 times, is placed in vent cabinet and is dried and keeps in Dark Place; 2) FA-PEG-The coupling of BSA: FA-PEG-NH25mg, EDC2.5mg, NHS2.5mg are dissolved in common room temperature in the BB that 500 μ LpH are 7Reaction 10min; Add BSA8.35mg, room temperature reaction 2h, after dialysis purifying 3 times in 4 DEG C of Refrigerator stores; 3) biotinylationThe preparation of BSA-FA: press long-chain biotin: incubated at room 30min after FA-PEG-BSA mol ratio 20:1 mixes, will mix moltenLiquid is in 4 DEG C of ultra-pure water dialysis 3d, and 4 DEG C save backup.
2 prepare the anti-sheep IgG of the anti-HE4 rabbit of biotinylation
1) getting biotin 15mg, EDC2.4mg, NHSS3.6mg, to be dissolved in 2mL0.02MpH be in 6.5PBS;2) add the anti-sheep IgG5.3mg of anti-HE4 rabbit, room temperature is placed in and on blending instrument, stirs 30min; 3) above-mentioned solution decompression is spin-dried for moltenAgent, deionized water dissolving, the 1d that dialyses in PBS and deionized water ,-20 DEG C of storages.
3 preparation SA-IO
1) 400 μ L5mg/mLIO are dissolved in the BB that 400 μ LpH are 5.5, according to IO:EDC mol ratio 1:1000, and IO:NHS mol ratio 1:2000 adds EDC/NHS, room temperature reaction 5 ~ 10min; 2) adjust pH value of solution to 8 ~ 8.5, add SA0.08Mg, continues to mix after 2h, adds the PBS sealing 10min cessation reaction of 20 μ L containing 5%BSA; 3) adding appropriate pH is 7BB mixes, and separation and purification IO-SA10 ~ 24h in 1.0 tesla's magnetic separators repeats to be resuspended in after 2 times 1mLpH and be 7In BB, new system magnetic bead concentration is 2mg/mL.
4 preparation SA-QDs
1) 100 μ LQDs(concentration are 8 μ M) be dissolved in the BB that 100 μ LpH are 5.5, according to QD:EDC mol ratio 1:1000, QD:NHS mol ratio 1:2000 adds EDC/NHS, room temperature reaction 5 ~ 10min; 2) adjust pH value of solution to 8 ~ 8.5, addSA1.056mg, continues to mix after 2h, adds the PBS sealing 10min cessation reaction of 10 μ L containing 5%BSA; 3) QD-SA is multipleCompound 10000r/min, centrifugal 5min removes after precipitation, uses the BB that 2mLpH is 7 to wash in 100K super filter tube,After to be resuspended in 0.1mLpH be in 7 BB.
5 magnetic nano-particle magnetic signal amplification systems based on biotin-SA and BAS mediation are in oophoroma separation of whole bloodThe application of circulating tumor cell
1) collect pathology clinically and be diagnosed as oophoroma and cancer patient's 20 examples and normal health health check-up female without chemotherapyProperty 100 Patients with Peripheral blood, the about 1mL of every routine sample left and right, each blood sample is preserved by the anticoagulant tube that is added with EDTA, blood sample is collecting 24In h, test to prevent lysis. Each blood sample derives from healthcare hospital for women & children of Jiangxi Province, Jiangxi Prov. Tumour Hospital, Jiangxi ProvinceFirst Affiliated Hospital Of Nanchang University and the second affiliated hospital of University Of Nanchang of Jiangxi Province. 2) according to biotinylation BSA-FA and FR moleIn blood sample, add biotinylation BSA-FA than 5:1, on blending instrument, room temperature mixes 1h, and the compound of formation is biotinylationBSA-FA-cell; 2) add IO-SA according to IO-SA and biotinylation BSA-FA mol ratio 4:1, continue to mix 30min, formCompound be IO-SA-biotinylation BSA-FA-cell; 3) IO-SA-biotinylation BSA-FA-cell complexes is placed inIn 1.0T magnetic separator, magnetic separates 1h, carefully abandons supernatant, is placed in 24 porocyte culture plates with 50 μ LPBS are resuspended.
The quantum dot immune fluorescent dyeing of 6 circulating tumor cells
1) adopt fixer fixed cell 15min at normal temperatures, use immediately cleaning solution rinsing 3 times; 2) use penetrating fluid normal temperatureLower permeation cell 20min, cleaning solution rinses the nonspecific binding site 1h using afterwards for 3 times on confining liquid closing cell; 3) removeConfining liquid, adds appropriate HE4 goat polyclonal antibody and cell to hatch altogether at normal temperatures 2h. Cleaning solution rinses 3 times (at every turn5min), add biotinylation-anti-HE4 rabbit anti-sheep IgG compound and cell to hatch altogether at normal temperatures 1h; 4) cleaning solution rinses 3Inferior (each 5min), adds appropriate SA-QDs compound reaction 30min, rinses and sloughs for 3 times after background colour in inversion fluorescence microscopyMicroscopic observation fluorescence signal also carries out quantitative analysis by fluorescence signal acquisition system.
Although the present invention discloses as above with preferred embodiment, so it is not in order to limit the present invention. Technology under anyThe technical staff in field, without departing from the spirit and scope of the present invention, when doing a little change and improvement, therefore the present inventionProtection domain be as the criterion when regarding the claim person of defining as.
Claims (8)
1. for the ovarian cancer cell detection kit of whole blood, it is characterized in that: described kit comprises 1) HE4 goat polycloneAntibody; 2) biotinylated BSA-FA; 3) the nanometer magnetic bead SA-IO of streptavidin; 4) QDs of streptavidinSA-QDs; 5) the anti-sheep IgG of the anti-HE4 rabbit of biotinylation; Described SA-QDs, its preparation method is as follows: 1) 100 μ L8 μ mol/LQDs is dissolved in the borate buffer that 100 μ LpH are 5.5, according to QDs:EDC mol ratio 1:1000, and QDs:NHS mol ratio 1:2000 adds EDC/NHS, room temperature reaction 5 ~ 10min; 2) adjust pH value of solution to 8 ~ 8.5, add SA1.056mg, continue to mix 2After h, add the PBS sealing 10min cessation reaction of 10 μ L containing 5%BSA; 3) SA-QDs compound 10000r/min, centrifugal 5Min removes after precipitation, uses the borate buffer that 2mLpH is 7 to wash in 100K super filter tube, is finally resuspended in 0.1mLPH is in 7 borate buffer.
2. kit according to claim 1, is characterized in that: described nanometer magnetic bead is Fe2O3And Fe3O4Compound is receivedRice magnetic bead, particle diameter is 25nm, magnetic bead surfaces is carboxyl-functional.
3. kit according to claim 1, is characterized in that: described biotinylation BSA-FA, its preparation method asUnder: 1) FA-PEG-NH2Coupling: FA6mg is dissolved in 600 μ L dimethyl sulfoxide (DMSO)s, adds DCC3mg, NHSS3mgAnd the common darkroom reaction of triethylamine 60 μ L 4h, add the reaction of two amino polyethylene glycol 30mg darkroom to spend the night; After reacting completely fromThe heart, uses-20 DEG C of acetone precipitations, and ethyl acetate washing 3 times, is placed in vent cabinet and is dried and keeps in Dark Place; 2) FA-PEG-BSACoupling: FA-PEG-NH25mg, EDC2.5mg, NHSS2.5mg are dissolved in the borate buffer that 500 μ LpH are 7 and are total toWith room temperature reaction 10min; Add BSA8.35mg, room temperature reaction 2h, after dialysis purifying 3 times in 4 DEG C of Refrigerator stores; 3) rawThe preparation of thing elementization BSA-FA: press long-chain biotin: incubated at room 30min after FA-PEG-BSA mol ratio 20:1 mixes, willMixed solution is in 4 DEG C of ultra-pure water dialysis 3d, and 4 DEG C save backup.
4. kit according to claim 1, is characterized in that: the anti-sheep IgG of the anti-HE4 rabbit of described biotinylation, its systemPreparation Method is as follows: 1) getting biotin 15mg, EDC2.4mg, NHSS3.6mg, to be dissolved in 2mL0.02MpH be 6.5 phosphoric acidIn salt buffer PBS; 2) add the anti-sheep IgG5.3mg of anti-HE4 rabbit, room temperature is placed in and on blending instrument, stirs 30min; 3) above-mentioned moltenLiquid decompression is spin-dried for solvent, deionized water dissolving, the 1d that dialyses in PBS and deionized water ,-20 DEG C of storages.
5. kit according to claim 1, is characterized in that: described SA-IO by SA and IO via covalent bond couplingPreparation; Its preparation method is as follows: 1) 400 μ L5mg/mLIO are dissolved in the borate buffer that 400 μ LpH are 5.5, according to IO:EDC mol ratio 1:1000, IO:NHS mol ratio 1:2000 adds EDC/NHS, room temperature reaction 5 ~ 10min; 2) adjust pH value of solutionTo 8 ~ 8.5, add SA0.08mg, continue to mix after 2h, add 20 μ L to stop anti-containing the PBS sealing 10min of 5%BSAShould; 3) adding appropriate pH is that 7 borate buffer mixes, separation and purification IO-SA10 ~ 24 in 1.0 tesla's magnetic separatorsH, repeats to be resuspended in after 2 times 1mLpH and is in 7 borate buffer, and new system magnetic bead concentration is 2mg/mL.
6. kit according to claim 1, is characterized in that: described kit also comprises fixer, penetrating fluid, sealingLiquid, cleaning solution.
7. kit according to claim 1, is characterized in that: described detection sample is whole blood.
8. kit according to claim 1, is characterized in that: described QDs is CdSe/ZnS, and emission wavelength is550nm。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410538494.9A CN104316683B (en) | 2014-10-14 | 2014-10-14 | For the ovarian cancer cell detection kit of whole blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410538494.9A CN104316683B (en) | 2014-10-14 | 2014-10-14 | For the ovarian cancer cell detection kit of whole blood |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104316683A CN104316683A (en) | 2015-01-28 |
| CN104316683B true CN104316683B (en) | 2016-05-11 |
Family
ID=52371941
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410538494.9A Active CN104316683B (en) | 2014-10-14 | 2014-10-14 | For the ovarian cancer cell detection kit of whole blood |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104316683B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106381286A (en) * | 2016-08-31 | 2017-02-08 | 上海美吉生物医药科技有限公司 | Folic acid immunomagnetic beads and preparation method thereof |
| CN107192823B (en) * | 2017-06-08 | 2024-03-19 | 杭州遂真生物技术有限公司 | Group B streptococcus enzyme-linked immunosorbent assay kit |
| CN108251415B (en) * | 2018-01-22 | 2020-05-15 | 北京中科圆融生物科技发展有限公司 | Anionic polypeptide carboxylated biological nano magnetic bead and preparation method thereof |
| CN108469519B (en) * | 2018-03-07 | 2020-03-24 | 深圳市伯劳特生物制品有限公司 | Composition for enzyme-linked immunosorbent assay kit, diabetes antibody spectrum detection kit and preparation method thereof |
| CN110501208B (en) * | 2018-05-17 | 2023-06-27 | 国家纳米科学中心 | Folic acid functionalized streptavidin modified magnetic nanoparticle, preparation method and application thereof |
| CN109406789A (en) * | 2018-11-23 | 2019-03-01 | 李翀 | A kind of bladder cancer circulating cells detection kit and application |
| CN110646382B (en) * | 2019-11-05 | 2020-10-16 | 上海大学 | High-sensitivity and double-selectivity CTC detection SPR sensor and preparation method thereof |
| CN111454907A (en) * | 2020-04-14 | 2020-07-28 | 厦门大学 | Circulating tumor cell rapid non-invasive capture, release and detection kit |
| CN111679086B (en) * | 2020-08-11 | 2020-11-13 | 苏州康和顺医疗技术有限公司 | HBP magnetic particle chemiluminescence detection kit and preparation method thereof |
| CN116120430A (en) * | 2023-02-27 | 2023-05-16 | 浙江准策生物技术有限公司 | Folic acid complete antigen and antibody, and preparation method and application thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7122384B2 (en) * | 2002-11-06 | 2006-10-17 | E. I. Du Pont De Nemours And Company | Resonant light scattering microparticle methods |
| CN101441218B (en) * | 2008-12-18 | 2013-10-09 | 东华大学 | Alpha fetal protein (AFP) detection method based on fluorescent nano luminous and magnetic nano material |
| CN102153149B (en) * | 2010-12-29 | 2012-10-24 | 济南大学 | Method for preparing nano quantum dot level Fe3O4 superparamagnetic particles |
| CN103275934A (en) * | 2013-06-05 | 2013-09-04 | 南昌大学 | Separation method of micro circulating tumor cells |
| CN103389381B (en) * | 2013-07-19 | 2015-07-15 | 武汉生之源生物科技有限公司 | Human epididymal secretory protein E4 chemiluminescence detection kit and preparation method thereof |
| CN103525414B (en) * | 2013-10-21 | 2016-02-17 | 北京理工大学 | Carbon quantum dot magnetic fluorescent dual-function nano material and preparation method thereof |
-
2014
- 2014-10-14 CN CN201410538494.9A patent/CN104316683B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104316683A (en) | 2015-01-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104316683B (en) | For the ovarian cancer cell detection kit of whole blood | |
| CN103756967B (en) | Application of the monoclonal antibody coupling immunomagnetic beads of anti-HLA-G in tumour cell sorting | |
| Song et al. | Fluorescent-magnetic-biotargeting multifunctional nanobioprobes for detecting and isolating multiple types of tumor cells | |
| Liu et al. | Folic acid conjugated magnetic iron oxide nanoparticles for nondestructive separation and detection of ovarian cancer cells from whole blood | |
| CN103472229B (en) | A kind of carcinomebryonic antigen magnetic microparticle chemiluminescence immune assay detection kit | |
| CN104316685B (en) | Diacetyl spermine detection kit and preparation method and application thereof | |
| CN106841613A (en) | A kind of method and system for detecting excretion body | |
| CN101520458B (en) | Simple method for detecting HLA-G and antibody thereof by gold-labeled immunoassay | |
| CN103923192B (en) | For polypeptide and the application thereof of enrichment, separation and detection circulating tumor cell | |
| CN104515859A (en) | Hemoglobin, hemoglobin-haptoglobin composite and transferrin joint examination kit and preparation method and detection method thereof | |
| CN106366196B (en) | EpCAM antibody immunomagnetic beads and preparation method thereof | |
| Li et al. | Affordable and simple method for separating and detecting ovarian cancer circulating tumor cells using BSA coated magnetic nanoprobes modified with folic acid | |
| CN103725648A (en) | Novel CTC (circulating tumor cell) enrichment technology and preparation method of kit | |
| CN103275934A (en) | Separation method of micro circulating tumor cells | |
| CN111454907A (en) | Circulating tumor cell rapid non-invasive capture, release and detection kit | |
| CN107271693A (en) | A kind of quick feces occult blood duplex inspects paper slip and preparation method thereof | |
| CN109856388A (en) | The catching method and capture kit of circulating tumor cell | |
| CN110456077A (en) | A method of the immunomagnetic beads enrichment detection CTCs of coupling anti-EpCAM antibody | |
| CN105588944A (en) | Application of AGP1, SERPINA3 and CDH1 content detection system in screening active tuberculosis patients | |
| CN110456076A (en) | The method of multiple antibody immune magnetic beads enrichment detection CTCs | |
| CN101320041B (en) | Colloidal gold method for fast quantitative determination of C-reaction protein and its application | |
| CN106366195B (en) | PD-L1 antibody immunomagnetic beads and preparation method thereof | |
| CN107525920B (en) | Poly ion liquid magnetic nanocomposites and its application to trace enriching specificity of circulating tumor cell and detection | |
| CN104990905A (en) | Kit for diagnosis of hepatocellular carcinoma metastasis based on solid-phase enzyme immunoassay fluorescence spots | |
| CN113588938B (en) | Immunomagnetic bead and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |