CN104316683A - Whole blood-oriented ovarian carcinoma cell detection kit - Google Patents
Whole blood-oriented ovarian carcinoma cell detection kit Download PDFInfo
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Abstract
The invention provides a whole blood-oriented ovarian carcinoma cell detection kit. The kit mainly comprises HE4 goat polyclonal antibodies capable of specifically recognizing the antigen of the ovarian carcinoma cell human epididymis protein (HE4), biotinylated bovine serum albumin (BSA)-folic acid (FA), streptavidin-modified nano magnetic beads (SA-IO), streptavidin-modified QDs (SA-QDs), biotinylated anti-HE4 rabbit anti-goat IgG, a connecting reagent and the like. The kit is capable of efficiently separating out the carcinoma cells in the whole blood of an ovarian carcinoma patient by virtue of the enrichment characteristic of the immunomagnetic beads, and realizing multiple-signal synergetic amplification by specially binding with the targeted ovarian carcinoma cells by use of the SA-QDs and a biotinylated HE4 antigen-antibody complex. The kit has high sensitivity and specificity, and has an extremely wide application prospect in early detection of the ovarian carcinoma.
Description
Technical field
The invention belongs to biotechnology and biomedical sector.Particularly, the invention provides a kind of kit detecting oophoroma.
Background technology
Oophoroma is gynaecology's common cancer, and its incidence of disease is arranged in feminine proses the 3rd, and case fatality rate but occupy the first.Be late period when most of oophoroma is made a definite diagnosis, its 5 years survival rates are only 30%, and early diagnosis and therapy oophoroma improves patient's prognosis, improves the key factor of its survival rate.At present, the screening technique that oophoroma is common clinically has 2 kinds: 1) Transvaginal Ultrasound inspection; 2) Serum tumor marker CA125 detects.All there is its limitation in the two.Research shows, in blood of cancer patients, the appearance of circulating tumor cell (CTCs) is early than visible solid tumor, and therefore, the method finding CTCs in a kind of high specific rich in human peripheral blood is the focus of research at present.
Immunity magnetic separation technique is one of important component part of CTCs fast separating concentration technology, the immunomagnetic beads that this technology relates to not only can binding active proteins matter or part but also can by attraction, through some process, after antibody or part are combined with magnetic bead, can be combined with target antigen specifically and form magnetic bead-antibody (part)-antigenic compound, then under the effect of externally-applied magnetic field by the object cell containing target antigen and other cell separation, thus reach the effect of separation and purification object cell.This technology has been widely used in diagnosis, the Treatment and Prognosis research of various tumour, but fresh few research in oophoroma early detection.Because folacin receptor (FR) is extremely low in most of normal tissue expression level, and at ovarian cancer cell surface expression high be 95%, FA and FR binding affinity be 0.19 nM, therefore, FA is connected to Fe by the present invention
2o
3and Fe
3o
4composite nanocrystalline surface, makes magnetic bead have targets identification and magnetic resolution dual-use function, thus realizes the enrichment of ovarian cancer cell, effectively improves detection sensitivity and realizes early detection oophoroma.
People 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%.To the evaluation study of the large number of biological mark that oophoroma is correlated with, one shows that HE4 detects and has the highest sensitivity in diagnosis of ovarian cancer in early days, its susceptibility is 82.7% and CA125 only has 45.9%.Therefore, HE4, as a kind of new tumor markers, can help early detection and the risk assessment of carrying out oophoroma.
Quantum dot (QDs) is nanocrystalline as a kind of novel fluorescence, has the fluorescence property of some uniquenesses, and as high in quantum yield, photochemical stability is high good, not easily photodissociation etc.At present QDs is combined the report preparing fluorescence immunoassay probe with antibody a lot, but still there is the problems such as signal is low, detection sensitivity is low when detecting very micro-material in QDs immune fluorescent probe.Biotin-avidin system is a kind of novel signal magnifying tags technology, and after utilizing Avidin to modify QDs, QDs can be combined with biotinylated antibody easily, effectively amplifies its fluorescence signal, thus realizes the detection of ultrasensitiveness ovarian cancer cell.
Summary of the invention
Key technical problem to be solved by this invention is to provide a kind of kit detecting ovarian cancer cell based on magnetic resolution and QDs immunofluorescence label for ovarian cancer cell quick detection kit in the whole blood that there is no at present.What utilize immunomagnetic beads can enriched character and the change of QDs fluorescence signal intensity, sets up that multiple signal is collaborative to be amplified, and has the kit detecting ovarian cancer cell in supersensitive whole blood fast.The resolution that the oophoroma that can improve this kit detects, sensitivity and specificity, more effectively carry out early detection and the risk assessment of oophoroma.
Ovarian cancer cell detection kit for whole blood disclosed by the invention comprises: HE4 goat polyclonal antibodies, biotinylated bovine serum albumin (BSA)-folic acid (FA), the nanometer magnetic bead (SA-IO) of streptavidin, the QDs(SA-QDs of streptavidin), the anti-sheep of biotinylation rabbit (HE4) IgG.
Described nanometer magnetic bead is Fe
2o
3and Fe
3o
4compound nanometer magnetic bead, particle diameter is 25nm, surface carboxyl functionalized.HE4 goat polyclonal antibodies and biotinylation BSA-FA respectively can the FR of HE4 antigen and surface of cell membrane in specific recognition ovarian cancer cell matter, wherein HE4 is process LAN antigen of (93%) on ovarian cancer cell matter golgiosome, and expression is lower in the normal tissue for it.The expression of FR on ovarian cancer cell surface is up to 95%, and extremely low in most of normal tissue expression amount.
Described SA-QDs, its preparation method is as follows: 1) 100 μ L, 8 μm of ol/L QDs are dissolved in 100 μ L pH is the BB of 5.5, adds EDC/NHS, room temperature reaction 5 ~ 10 min according to QD:EDC mol ratio 1:1 000, QD:NHS mol ratio 1:2 000; 2) adjust pH value of solution to 8 ~ 8.5, add SA 1.056 mg, after continuing mixing 2 h, add the PBS closed 10 min cessation reactions of 10 μ L containing 5% BSA; 3) SA-QDs compound 10 000 r/min, centrifugal 5 min removing precipitation after, use 2 mL pH be 7 BB wash in 100K super filter tube, being finally resuspended in 0.1 mL pH is in the BB of 7.
Described biotinylation BSA-FA, its preparation method is as follows: 1) FA-PEG-NH
2coupling: FA 6 mg is dissolved in 600 μ L dimethyl sulfoxide (DMSO)s, add N, N-dicyclohexylcarbodiimide (DCC) 3 mg, N, 4 h are reacted in N-hydroxy-succinamide (NHSS) 3 mg and the common darkroom of triethylamine 60 μ L, add the reaction of two amino-polyethyleneglycols 30 mg darkrooms and spend the night; Centrifugal after reacting completely, use-20 DEG C of acetone precipitations, ethyl acetate washs 3 times, is placed in vent cabinet drying and keeps in Dark Place; 2) coupling of FA-PEG-BSA: FA-PEG-NH
25 mg, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) 2.5 mg, NHSS 2.5 mg to be dissolved in 500 μ L pH be common room temperature reaction 10 min in the borate buffer (BB) of 7; Add BSA 8.35 mg, room temperature reaction 2 h, in 4 DEG C of Refrigerator stores after dialysis purifying 3 times; 3) preparation of biotinylation BSA-FA: by long-chain biological element: incubated at room 30 min after FA-PEG-BSA mol ratio 20:1 mixes, to be dialysed 3 d in 4 DEG C of ultrapure waters by mixed solution, 4 DEG C save backup.
Described biotinylation BSA-FA mainly utilizes amino reactive group abundant on large molecule BSA, volume biotin and FA is enriched on BSA, thus increase FA realizes amplifying in conjunction with quantity with IO-SA with Cell binding probability and biotin.
The anti-sheep IgG of described biotinylation anti-HE4 rabbit, its preparation method is as follows: 1) getting biotin 15 mg, EDC 2.4mg, NHSS 3.6mg, to be dissolved in 2 mL 0.02 M pH be in 6.5 phosphate buffers (PBS); 2) add anti-HE4 rabbit anti-sheep IgG 5.3 mg, room temperature is placed on blending instrument and stirs 30 min; 3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d 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 L 5mg/mL IO are dissolved in 400 μ L pH is the BB of 5.5, adds EDC/NHS, room temperature reaction 5 ~ 10 min according to IO:EDC mol ratio 1:1 000, IO:NHS mol ratio 1:2 000; 2) adjust pH value of solution to 8 ~ 8.5, add SA 0.08 mg, after continuing mixing 2 h, add the PBS closed 10 min cessation reactions of 20 μ L containing 5% BSA; 3) add the BB mixing that appropriate pH is 7, separation and purification IO-SA 10 ~ 24 h in 1.0 tesla's magnetic separators, being resuspended in 1 mL pH after repeating 2 times is in the BB of 7, and brand-new magnetic bead concentration is 2 mg/mL.Wherein, 5%BSA/PBS is combined by the carboxyl of IO or the QDs remained on surface in coupled product of the active amino on BSA, reduces the non-specific adsorption of coupled product.
Described kit also comprises immobile liquid (4% paraformaldehyde/trihydroxy aminomethane-hydrochloride buffer (TBS) pH 7.6), penetrating fluid (TBS+0.1% Tween-20 pH 7.6), confining liquid (0.1% casein+5%BSA+TBS pH 7.6), cleansing solution (TBS pH 7.6).
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 carry out fluorescence labeling by the QDs of specific antibody modificationization, observe counting separation efficiency and fluorescence signal acquisition systematic quantification analysis acquisition final detection result by inverted fluorescence microscope.
Beneficial effect: oophoroma case fatality rate in gynecologic malignant tumor is in first, set up easy, ovarian cancer cell detection method is not only to the early detection of tumour fast, also has great importance to the rapid evaluation of Chemotherapy of Tumor Patients medicine, individualized treatment (detection of clinical sieve medicine, drug resistance), the monitoring of tumor recurrence and the exploitation of tumour new drug.
The present invention, by antibody molecule and biotin coupling, avoids in conventional method and antibody molecule is coupled to QDs or magnetic bead surfaces and causes antibody activity to reduce and sterically hindered large shortcoming.
The present invention utilizes amino reactive group abundant on large molecule BSA, volume biotin and FA is enriched on BSA, thus increase FA realizes amplifying in conjunction with quantity with IO-SA with Cell binding probability and biotin.
The present invention is according to the double-antibody sandwich principle of fluorescence immunoassay, utilize the advantages such as immuno magnetic cell separation speed is fast, efficiency is high, easy and simple to handle, high in conjunction with QDs photochemical stability, the optical characteristics that not easily photodissociation etc. are excellent, respectively with immunomagnetic beads and QDs for carrier is connected biotinylated BSA-FA and the anti-sheep IgG of anti-HE4 rabbit, the detection of cancer cell in whole blood is quickly and accurately achieved.
Accompanying drawing explanation
The SKOV3 cell of Fig. 1 based on magnetic resolution and the separation efficiency figure of A549 cell, the separation efficiency of SKOV3 cell when wherein SKOV3-2 is the simple IO-SA Magneto separate not adding biotinylation BSA-FA reaction.
The QDs immunofluorescence label coloration result of Fig. 2 SKOV3 cell and A549 cell.
Embodiment
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 used in embodiment and QD550 purchased from American Ocean NanoTech company, biotin, SA are purchased from Shanghai Chinese blue chemical company.HE4 goat polyclonal antibodies (C-12) is purchased from Santa Cruz biotech company, the anti-sheep of rabbit (HE4) IgG(BA1040) be purchased from doctor's moral biotech company, DCC, NHSS, NHS, EDC buy in Sigma-Aldrich, BSA purchased from Biosharp company, folic acid purchased from
zhengzhou Song Hua commerce and trade company limited, inverted fluorescence microscope is Japanese Nikon Products, and the analysis of fluorescence signal acquisition systematic quantification is online download ImageJ 2x software analysis gained.
Nanometer magnetic bead is Fe
2o
3and Fe
3o
4compound nanometer magnetic bead, particle diameter is 25nm, surface carboxyl functionalized.
Described QDs is CdSSe/ZnS, and emission wavelength is 550nm.
Write a Chinese character in simplified form in embodiment:
The nanometer magnetic bead (SA-IO) of quantum dot (QDs), ovarian cancer cell people epididymal proteins (HE4), bovine serum albumin (BSA), folic acid (FA), streptavidin, the QDs(SA-QDs of streptavidin), folacin receptor (FR).
Immobile liquid (4% paraformaldehyde/trihydroxy aminomethane-hydrochloride buffer (TBS) pH 7.6), penetrating fluid (TBS+0.1% Tween-20 pH 7.6), confining liquid (0.1% casein+5%BSA+TBS pH 7.6), cleansing solution (TBS pH 7.6).
Embodiment 1 detects oophoroma SKOV3 cell for the ovarian cancer cell detection kit of whole blood
1 prepares biotinylation BSA-FA
1) FA-PEG-NH
2coupling: FA 6 mg is dissolved in 600 μ L dimethyl sulfoxide (DMSO)s, add DCC 3 mg, NHSS 3 mg and the common darkroom of triethylamine 60 μ L react 4 h, add the reaction of two amino-polyethyleneglycols 30 mg darkrooms and spend the night; Centrifugal after reacting completely, use-20 DEG C of acetone precipitations, ethyl acetate washs 3 times, is placed in vent cabinet drying and keeps in Dark Place; 2) coupling of FA-PEG-BSA: FA-PEG-NH
25 mg, EDC 2.5 mg, NHS 2.5 mg to be dissolved in 500 μ L pH be common room temperature reaction 10 min in the BB of 7; Add BSA 8.35 mg, room temperature reaction 2 h, in 4 DEG C of Refrigerator stores after dialysis purifying 3 times; 3) preparation of biotinylation BSA-FA: by long-chain biological element: incubated at room 30 min after FA-PEG-BSA mol ratio 20:1 mixes, to be dialysed 3 d in 4 DEG C of ultrapure waters by mixed solution, 4 DEG C save backup.
2 prepare the anti-sheep IgG of biotinylation anti-HE4 rabbit
1) getting biotin 15 mg, EDC 2.4mg, NHSS 3.6mg, to be dissolved in 2 mL 0.02 M pH be in 6.5 PBS; 2) add anti-HE4 rabbit anti-sheep IgG 5.3 mg, room temperature is placed on blending instrument and stirs 30 min; 3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water ,-20 DEG C of storages.
3 preparation SA-IO
1) 400 μ L IO(concentration are 5mg/mL) to be dissolved in 400 μ L pH be the BB of 5.5, adds EDC/NHS, room temperature reaction 5 ~ 10 min according to IO:EDC mol ratio 1:1 000, IO:NHS mol ratio 1:2 000; 2) adjust pH value of solution to 8 ~ 8.5, add SA 0.08 mg, after continuing mixing 2 h, add the PBS closed 10 min cessation reactions of 20 μ L containing 5% BSA; 3) add the BB mixing that appropriate pH is 7, separation and purification IO-SA 10 ~ 24 h in 1.0 tesla's magnetic separators, being resuspended in 1 mL pH after repeating 2 times is in the BB of 7, and brand-new magnetic bead concentration is 2 mg/mL.
4 preparation SA-QDs
1) 100 μ L QDs(concentration are 8 μMs) to be dissolved in 100 μ L pH be the BB of 5.5, adds EDC/NHS, room temperature reaction 5 ~ 10 min according to QD:EDC mol ratio 1:1 000, QD:NHS mol ratio 1:2 000.2) adjust pH value of solution to 8 ~ 8.5, add SA 1.056 mg, after continuing mixing 2 h, add the PBS closed 10 min cessation reactions of 10 μ L containing 5% BSA.3) QD-SA compound 10 000 r/min, centrifugal 5 min removing precipitation after, use 2 mL pH be 7 BB wash in 100K super filter tube, being finally resuspended in 0.1 mL pH is in the BB of 7.
The IO(IO-FA of 5 preparation FA functionalized modifications)
1) FA-PEG-NH
2coupling: FA 6 mg is dissolved in 600 μ L dimethyl sulfoxide (DMSO)s, add DCC 3 mg, NHSS 3 mg and the common darkroom of triethylamine 60 μ L react 4 h, add the reaction of two amino-polyethyleneglycols 30 mg darkrooms and spend the night; 2) IO and FA-PEG-NH
2coupling: 200 μ L IO(concentration are 5mg/mL) to be dissolved in 200 μ L pH be the BB of 5.5, adds EDC/NHS, room temperature reaction 5 ~ 10 min according to IO:EDC mol ratio 1:1 000, IO:NHS mol ratio 1:2 000.2) adding 500 μ L pH is 8 ~ 8.5 BB, adds FA-PEG-NH
27 mg, after continuing mixing 2 h, add the PBS closed 10 min cessation reactions of 10 μ L containing 5% BSA; 3) add the BB mixing that appropriate pH is 7, separation and purification IO-FA 10 ~ 24 h in 1.0 tesla's magnetic separators, being resuspended in 500 μ L pH after repeating 2 times is in the BB of 7, and brand-new magnetic bead concentration is 2 mg/mL.
The dyeing of 6 SKOV3 cells and counting
1) obtain oophoroma SKOV3 cell 4 mL of exponential phase, add 8 μ L Hoechest core dyestuffs, be placed in 37 DEG C, hatch 30 min in 5% CO2 incubator; 1 000 r/min, abandon supernatant after centrifugal 30 s, add PBS 10 mL and clean, 1 000 r/min 1 min(repeated washing 2 times); Resuspended with 4 mL 3%BSA/PBS.2) by the quantity of cell counting count board counting (the little lattice of 25 large lattice * 16) SKOV3 cell, counting principle under fluorescent microscope is: under not several on several right side, a line ball cell number left side, number.
7 IO-FA magnetic nano-particle separation of whole blood SKOV3 cells
1) according to 1:10
9prestained SKOV3 cell adds in 1 mL whole blood by/mL, after room temperature fully mixes 1 h on blending instrument, adds IO-FA magnetic nano-particle according to IO-FA and FR mol ratio 20:1, continues mixing 30 min, forms composite I O-FA-cell; 2) IO-FA-cell complexes is placed in 1.0T magnetic separator Magneto separate 1 h, carefully abandons supernatant, be placed in 24 porocyte culture plates with 50 μ L PBS are resuspended.
The 8 magnetic nano-particle magnetic signal amplification separation of whole blood SKOV3 cells mediated based on biotin-SA and BAS
1) according to 1:10
9prestained SKOV3 cell adds in 1 mL whole blood by/mL, and fully after mixing, be that 5:1 adds biotinylation BSA-FA according to biotinylation BSA-FA and FR mol ratio, on blending instrument, room temperature mixes 1 h, and the compound of formation is biotinylation BSA-FA-cell; 2) add IO-SA according to IO-SA and biotinylation BSA-FA mol ratio 4:1, continue mixing 30 min, the compound of formation is IO-SA-biotinylation BSA-FA-cell; 3) IO-SA-biotinylation BSA-FA-cell complexes is placed in 1.0T magnetic separator Magneto separate 1 h, carefully abandons supernatant, be placed in 24 porocyte culture plates with 50 μ L PBS are resuspended.
The quantum dot immune fluorescent dyeing of 9 SKOV3 cells
1) adopt immobile liquid fixed cell 15min at normal temperatures, use cleansing solution rinsing immediately 3 times; 2) with permeation cell 20min under penetrating fluid normal temperature, cleansing solution rinses the nonspecific binding site 1h used afterwards for 3 times on confining liquid closing cell; 3) removing confining liquid, adds appropriate HE4 goat polyclonal antibodies and cell hatches 2h at normal temperatures altogether.After cleansing solution rinses 3 times (each 5min), add biotinylation-anti-HE4 rabbit anti-sheep IgG compound and cell hatches 1h at normal temperatures altogether; 4) cleansing solution rinses 3 times (each 5min), adds appropriate SA-QDs compound reaction 30min, rinses and under inverted fluorescence microscope, observes fluorescence signal after sloughing background colour 3 times and carry out quantitative test and calculating separation efficiency by fluorescence signal acquisition system.
10 magnetic bead non specific control experiments
1) select SKOV3 cell, step 1-4 is the same; 2) cell count is with step 6, and SKOV3 cellular whole blood Magneto separate process does not add biotinylation BSA-FA, and all the other operation stepss are with 8,9.
11 QDs non specific control experiments
1) select SKOV3 cell, step 1-8 is the same.2) do not add HE4 goat polyclonal antibodies and biotinylation-anti-goat-anti nanocrystal composition of anti-HE4 rabbit in the quantum dot immune fluorescent dyeing course of the SKOV3 cell after whole blood Magneto separate, all the other steps are with 9.
12 concurrent control group experiments
FR is selected to express lung cancer A549 cell that is negative and the low expression of HE4, the same 1-9 of all the other operation stepss.
(above-mentioned steps 7,8,10,12 is the experiment simultaneously carried out under equal conditions, and experiment number is no less than 3 times; Step 9 and 11 is the identification experiment simultaneously carried out)
11 results:
1) Hoechst core dyestuff can fully locate oophoroma SKOV3 cell, via the SKOV3 that biotinylation-BSA-FA/IO-SA and IO-FA immunity Magneto separate and the anti-sheep Antibody-antigen complex of QDs-biotinylation-anti-HE4 rabbit mark, cell number is all more, wherein, biotinylation BSA-FA/IO-SA can up to 87.1% through many experiments to the separation efficiency of SKOV3 cell, and the separation efficiency of IO-FA is up to 43%, under equivalent responses condition, A549 cell capture number is rare, and its capture rate only has 23.3% and 23.1%.In addition, the non-specific experiment of magnetic bead shows, when not having FA to participate in, the separation efficiency of IO-SA is all not high, and it is about about 8.7% to the separation efficiency of SKOV3 cell.And to the separation efficiency of A549 cell also at about 8.6% (Fig. 1).Result shows, FA can specific recognition oophoroma SKOV3 cell, and the magnetic nano-particle magnetic signal amplification system through biotin-SA and BAS mediation can significantly improve cell capture efficiency, improves cell detection sensitivity.(Fig. 1)
2) via the SKOV3 cell that biotinylation-BSA-FA/IO-SA and IO-FA immunity Magneto separate and the anti-sheep Antibody-antigen complex of QDs-biotinylation-anti-HE4 rabbit mark, all visible significantly green fluorescence in tenuigenin, and the A549 cell that Magneto separate is caught has no obvious green fluorescence after mark, do not add HE4 goat polyclonal antibodies and biotinylation-anti-HE4 rabbit anti-goat-anti nanocrystal composition by the SKOV3 cell of the non-specific mark of SA-QDs, in tenuigenin, have no obvious green fluorescence.(Fig. 2)
3) in clinical practice sample, 20 routine ovarian cancer patients peripheral bloods and 100 routine healthy women peripheral bloods show after using this kit to detect, find circulating tumor cell in 16 routine ovarian cancer patients peripheral bloods, and do not find circulating tumor cell in healthy women peripheral blood.(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 | Borderline 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 | Borderline MCAC | 1 | 0 |
| 15 | Borderline 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 |
More than test the immunofluorescence label demonstrating magnetic resolution and the anti-HE4 antigen antibody complex of QDs-SA/ biotinylation that the present invention is based on biotinylation BSA-FA/IO-SA can be used for specific enrichment oophoroma SKOV3 cell and carry out specificity verification to it, thus can be used for the detection of early ovarian cancer.It is after the positive that the inventive method detects oophoroma result, can further adopt other technologies means to make a definite diagnosis oophoroma.
Embodiment 2 is for the application of ovarian cancer cell detection kit in ovarian cancer patients whole blood of whole blood
Get kit of the present invention: comprise 1) HE4 goat polyclonal antibodies; 2) biotinylated BSA-FA; 3) the nanometer magnetic bead SA-IO of streptavidin; 4) QDs and SA-QDs of streptavidin; 5) the anti-sheep IgG of biotinylation anti-HE4 rabbit;
Wherein, each substances preparation method is as follows:
1 prepares biotinylation BSA-FA
1) FA-PEG-NH
2coupling: FA 6 mg is dissolved in 600 μ L dimethyl sulfoxide (DMSO)s, add DCC 3 mg, NHS 3 mg and the common darkroom of triethylamine 60 μ L react 4 h, add the reaction of two amino-polyethyleneglycols 30 mg darkrooms and spend the night; Centrifugal after reacting completely, use-20 DEG C of acetone precipitations, ethyl acetate washs 3 times, is placed in vent cabinet drying and keeps in Dark Place; 2) coupling of FA-PEG-BSA: FA-PEG-NH
25 mg, EDC 2.5 mg, NHS 2.5 mg to be dissolved in 500 μ L pH be common room temperature reaction 10 min in the BB of 7; Add BSA 8.35 mg, room temperature reaction 2 h, in 4 DEG C of Refrigerator stores after dialysis purifying 3 times; 3) preparation of biotinylation BSA-FA: by long-chain biological element: incubated at room 30 min after FA-PEG-BSA mol ratio 20:1 mixes, to be dialysed 3 d in 4 DEG C of ultrapure waters by mixed solution, 4 DEG C save backup.
2 prepare the anti-sheep IgG of biotinylation anti-HE4 rabbit
1) getting biotin 15 mg, EDC 2.4mg, NHSS 3.6mg, to be dissolved in 2 mL 0.02 M pH be in 6.5 PBS; 2) add anti-HE4 rabbit anti-sheep IgG 5.3 mg, room temperature is placed on blending instrument and stirs 30 min; 3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water ,-20 DEG C of storages.
3 preparation SA-IO
1) 400 μ L 5mg/mL IO are dissolved in 400 μ L pH is the BB of 5.5, adds EDC/NHS, room temperature reaction 5 ~ 10 min according to IO:EDC mol ratio 1:1 000, IO:NHS mol ratio 1:2 000; 2) adjust pH value of solution to 8 ~ 8.5, add SA 0.08 mg, after continuing mixing 2 h, add the PBS closed 10 min cessation reactions of 20 μ L containing 5% BSA; 3) add the BB mixing that appropriate pH is 7, separation and purification IO-SA 10 ~ 24 h in 1.0 tesla's magnetic separators, being resuspended in 1 mL pH after repeating 2 times is in the BB of 7, and brand-new magnetic bead concentration is 2 mg/mL.
4 preparation SA-QDs
1) 100 μ L QDs(concentration are 8 μMs) to be dissolved in 100 μ L pH be the BB of 5.5, adds EDC/NHS, room temperature reaction 5 ~ 10 min according to QD:EDC mol ratio 1:1 000, QD:NHS mol ratio 1:2 000; 2) adjust pH value of solution to 8 ~ 8.5, add SA 1.056 mg, after continuing mixing 2 h, add the PBS closed 10 min cessation reactions of 10 μ L containing 5% BSA; 3) QD-SA compound 10 000 r/min, centrifugal 5 min removing precipitation after, use 2 mL pH be 7 BB wash in 100K super filter tube, being finally resuspended in 0.1 mL pH is in the BB of 7.
5 based on the magnetic nano-particle magnetic signal amplification system of biotin-SA and BAS mediation in the application of oophoroma separation of whole blood circulating tumor cell
1) collect definitive pathological diagnosis be clinically oophoroma and without chemotherapy cancer patient 20 example and normal health health check-up women 100 Patients with Peripheral blood, every routine sample about 1 about mL, each blood sample is preserved by the anticoagulant tube being added with EDTA, and blood sample carries out testing to prevent lysis in collection 24 h.Each blood sample derives from healthcare hospital for women & children of Jiangxi Province, Jiangxi Prov. Tumour Hospital, First Affiliated Hospital Of Nanchang University of Jiangxi Province and affiliated hospital of University Of Nanchang of Jiangxi Province second.2) in blood sample, add biotinylation BSA-FA according to biotinylation BSA-FA and FR mol ratio 5:1, on blending instrument, room temperature mixes 1 h, and the compound of formation is biotinylation BSA-FA-cell; 2) add IO-SA according to IO-SA and biotinylation BSA-FA mol ratio 4:1, continue mixing 30 min, the compound of formation is IO-SA-biotinylation BSA-FA-cell; 3) IO-SA-biotinylation BSA-FA-cell complexes is placed in 1.0T magnetic separator Magneto separate 1 h, carefully abandons supernatant, be placed in 24 porocyte culture plates with 50 μ L PBS are resuspended.
The quantum dot immune fluorescent dyeing of 6 circulating tumor cells
1) adopt immobile liquid fixed cell 15min at normal temperatures, use cleansing solution rinsing immediately 3 times; 2) with permeation cell 20min under penetrating fluid normal temperature, cleansing solution rinses the nonspecific binding site 1h used afterwards for 3 times on confining liquid closing cell; 3) removing confining liquid, adds appropriate HE4 goat polyclonal antibodies and cell hatches 2h at normal temperatures altogether.After cleansing solution rinses 3 times (each 5min), add biotinylation-anti-HE4 rabbit anti-sheep IgG compound and cell hatches 1h at normal temperatures altogether; 4) cleansing solution rinses 3 times (each 5min), adds appropriate SA-QDs compound reaction 30min, rinses and under inverted fluorescence microscope, observe fluorescence signal after sloughing background colour 3 times and carry out quantitative test by fluorescence signal acquisition system.
Although the present invention with preferred embodiment disclose as above, so itself and be not used to limit the present invention.Any person of ordinary skill in the field, without departing from the spirit and scope of the present invention, when doing a little change and improvement, therefore protection scope of the present invention is as the criterion when regarding as the claim person of defining.
Claims (8)
1. for the ovarian cancer cell detection kit of whole blood, it is characterized in that: described kit comprises 1) HE4 goat polyclonal antibodies; 2) biotinylated BSA-FA; 3) the nanometer magnetic bead SA-IO of streptavidin; 4) QDs and SA-QDs of streptavidin; 5) the anti-sheep IgG of biotinylation anti-HE4 rabbit; Described SA-QDs, its preparation method is as follows: 1) 100 μ L, 8 μm of ol/L QDs are dissolved in 100 μ L pH is the BB of 5.5, adds EDC/NHS, room temperature reaction 5 ~ 10 min according to QD:EDC mol ratio 1:1 000, QD:NHS mol ratio 1:2 000; 2) adjust pH value of solution to 8 ~ 8.5, add SA 1.056 mg, after continuing mixing 2 h, add the PBS closed 10 min cessation reactions of 10 μ L containing 5% BSA; 3) SA-QDs compound 10 000 r/min, centrifugal 5 min removing precipitation after, use 2 mL pH be 7 BB wash in 100K super filter tube, being finally resuspended in 0.1 mL pH is in the BB of 7.
2. kit according to claim 1, is characterized in that: described nanometer magnetic bead is Fe
2o
3and Fe
3o
4compound nanometer magnetic bead, particle diameter is 25nm, and magnetic bead surfaces is carboxyl-functional.
3. kit according to claim 1, is characterized in that: described biotinylation BSA-FA, and its preparation method is as follows: 1) FA-PEG-NH
2coupling: FA 6 mg is dissolved in 600 μ L dimethyl sulfoxide (DMSO)s, add N, N-dicyclohexylcarbodiimide (DCC) 3 mg, N, 4 h are reacted in N-hydroxy-succinamide (NHSS) 3 mg and the common darkroom of triethylamine 60 μ L, add the reaction of two amino-polyethyleneglycols 30 mg darkrooms and spend the night; Centrifugal after reacting completely, use-20 DEG C of acetone precipitations, ethyl acetate washs 3 times, is placed in vent cabinet drying and keeps in Dark Place; 2) coupling of FA-PEG-BSA: FA-PEG-NH
25 mg, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) 2.5 mg, NHSS 2.5 mg to be dissolved in 500 μ L pH be common room temperature reaction 10 min in the borate buffer (BB) of 7; Add BSA 8.35 mg, room temperature reaction 2 h, in 4 DEG C of Refrigerator stores after dialysis purifying 3 times; 3) preparation of biotinylation BSA-FA: by long-chain biological element: incubated at room 30 min after FA-PEG-BSA mol ratio 20:1 mixes, to be dialysed 3 d in 4 DEG C of ultrapure waters by mixed solution, 4 DEG C save backup.
4. kit according to claim 1, it is characterized in that: the anti-sheep IgG of described biotinylation anti-HE4 rabbit, its preparation method is as follows: 1) getting biotin 15 mg, EDC 2.4mg, NHSS 3.6mg, to be dissolved in 2 mL 0.02 M pH be in 6.5 phosphate buffers (PBS); 2) add anti-HE4 rabbit anti-sheep IgG 5.3 mg, room temperature is placed on blending instrument and stirs 30 min; 3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water ,-20 DEG C of storages.
5. kit according to claim 1, is characterized in that: described SA-IO is prepared via covalent bond coupling by SA and IO; Its preparation method is as follows: 1) 400 μ L 5mg/mL IO are dissolved in 400 μ L pH is the BB of 5.5, adds EDC/NHS, room temperature reaction 5 ~ 10 min according to IO:EDC mol ratio 1:1 000, IO:NHS mol ratio 1:2 000; 2) adjust pH value of solution to 8 ~ 8.5, add SA 0.08 mg, after continuing mixing 2 h, add the PBS closed 10 min cessation reactions of 20 μ L containing 5% BSA; 3) add the BB mixing that appropriate pH is 7, separation and purification IO-SA 10 ~ 24 h in 1.0 tesla's magnetic separators, being resuspended in 1 mL pH after repeating 2 times is in the BB of 7, and brand-new magnetic bead concentration is 2 mg/mL.
6. kit according to claim 1, is characterized in that: described kit also comprises immobile liquid, penetrating fluid, confining liquid, cleansing 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 CdSSe/ZnS, and emission wavelength is 550nm.
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