CN109975554A - The detection method and its special agent of cell PD-L1 protein expression in a kind of body fluid sample - Google Patents
The detection method and its special agent of cell PD-L1 protein expression in a kind of body fluid sample Download PDFInfo
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Abstract
The invention discloses the detection methods and its special agent of cell PD-L1 protein expression in a kind of body fluid sample, feature is the following steps are included: by the immune magnetic particle mixed liquor of preparation in conjunction with neoplasm circulating cells in body fluid sample, it is subsequently placed in immune magnetic particle capture instrument and is captured, the tumour cell of capture is dyed by PD-L1 immunohistochemical staining reagent, assess the protein expression of PD-L1, it is that magnetic particle is immunized in coating anti-human epcam that magnetic particle mixed liquor, which is wherein immunized, magnetic particle is immunized in anti-human ICAM-1, anti-human EGFR antibody mediated immunity magnetic particle, magnetic particle is immunized in anti-human folacin receptor, magnetic particle is immunized in anti-human CD10, magnetic particle is immunized in anti human CD 19, magnetic particle is immunized in anti-humen CD 20 and at least one of magnetic particle is immunized in anti-human CD33, advantage is tool There are easy to operate, high sensitivity and the easy interpretation of testing result.
Description
Technical field
The invention belongs to the immunohistochemistry detection fields of albumen, more particularly, to cell PD-L1 egg in a kind of body fluid sample
The detection method and its special agent of white expression.
Background technique
PD-1 is I type cross-film immunoglobulin receptor, belongs to the immune response superfamily member of CD28 mediation, is T cell
The important inhibition molecule on surface, intracellular section is converted motif (ITSM) containing ITIM and immunity receptor tyrosine,
ITSM has mediated the recruitment of Protein-tyrosine-phosphatase family phosphatase and the inhibition to T cell activation signal.PD-1 by
Body includes PD-L1(B7-H1) and PD-L2(B7-DC), important work is mainly played in the tumor microenvironment of immune system effector phase
With.PD-L1 has the expression of inductivity in the hematopoietic cell of kinds of tumor cells and tumor microenvironment, passes through the knot with PD-1
Close, induction generate body immunosupress, thus make tumour cell adaptive immune escape, this may is that tumour tolerance mechanism it
One.If being incorporated into blocking, it is possible to block this immunosupress, make the T cell activation of body itself, exempt to reach
The purpose of epidemic disease treatment.
PD-L1 inhibitor can block the combination of PD-1 and PD-L1, and tumour cell is made " to lighten restrictions on " T cell, and recovery is swollen
The immune function that oncocyte weakens.PD-L1 inhibitor treats effective and Small side effects to kinds of tumors, but it is not " omnipotent yet
Medicine " causes drug effect also different because of individual difference, and objective remission rate is used alone and only has 15-30%.It is for judgement inhibitor
It is no effective to patient, need to carry out the relevant detection of PD-L1.Currently, there are no single indexs clearly to accuse in the market
Tell whether patient's drug is effective, but can detecte the antibody expression of PD-L1 to carry out assessment drug effect.PD-L1 antibody
Detection is mainly based upon the detection of cell protein level, in clinical test based on ImmunohistochemistryMethods Methods, i.e., to operation or puncture
The tumor tissues obtained afterwards be sliced-dye and (pass through antibody), according to the coloring depth come evaluation expression situation.To being at present
Only, ImmunohistochemistryMethods Methods (for detecting PD-L1 antibody) be suitable for tissue, not yet refer to whether can be used for Pleural effusions, marrow and
The PD-L1 protein expression of the tumour cells such as peripheral blood detects.
Summary of the invention
Technical problem to be solved by the invention is to provide one kind, and there is easy to operate, high sensitivity and testing result easily to sentence
The detection method of cell PD-L1 protein expression in the body fluid sample of reading.
The technical scheme of the invention to solve the technical problem is:
1, in a kind of body fluid sample cell PD-L1 protein expression detection method, comprising the following steps: the immune magnetic of preparation is micro-
Grain mixed liquor is subsequently placed in immune magnetic particle capture instrument and is captured, will be caught in conjunction with neoplasm circulating cells in body fluid sample
The tumour cell obtained is dyed by PD-L1 immunohistochemical staining reagent, assesses the protein expression of PD-L1.
Magnetic particle is immunized for coating anti-human epcam in the immune magnetic particle mixed liquor, magnetic particle is immunized in anti-human ICAM-1,
Magnetic particle is immunized in anti-human EGFR antibody mediated immunity magnetic particle, anti-human folacin receptor, magnetic particle is immunized in anti-human CD10, anti human CD 19 is immunized
Magnetic particle is immunized in magnetic particle, anti-humen CD 20 and at least one of magnetic particle is immunized in anti-human CD33.
Specific step is as follows for the preparation method of the immune magnetic particle:
(1) pretreatment of magnetic particle: 0.05mol/L MES buffer is added in the centrifuge tube containing magnetic particle, by centrifuge tube
It is placed on magnetic frame after stratification, inhales and abandon supernatant, the 0.05mol/L MES buffer of equivalent is added into centrifuge tube, carry out
It after ultrasonic treatment, is placed on magnetic frame after stratification, inhales and abandon supernatant, repeat the above steps 2-4 times；Add again in centrifuge tube
Enter 0.5mL 0.05mol/L MES buffer, continuation is ultrasonically treated in Ultrasound Instrument, obtains that in the same size, shape is complete
And the magnetic particle suspension being evenly distributed；Wherein additive amount of MES buffer is that every 2mg magnetic particle addition 1mL MES is slow
Fliud flushing；Magnetic particle is the brown round or ellipse magnetic particle of partial size 200-1000nm, and carboxyl-content is 200 μm of ol/g-600 μ
mol/g；
(2) magnetic particle activates: 50mg/mL NHS solution activator and 50mg/mL EDAC are separately added into magnetic particle suspension
Solution activator is subsequently placed on adjustable rotary mixer, and after 25 ~ 40rpm, 37 ± 2 DEG C of reaction 30min, HEPS is added
Buffer carries out rinse, is placed on magnetic frame and washes repeatedly 2-4 times, inhales and abandons magnetic particle after supernatant is activated；Wherein NHS is molten
The additive amount of liquid, EDAC solution and HEPS buffer be every 2mg magnetic particle add 100 μ LNHS solution, 100 μ LEDAC solution and
1mL HEPS buffer；
(3) magnetic particle coupled antibody: after antibody sealing is added in the centrifuge tube of magnetic particle after containing activation, it is put into vertical rotary
On mixer, in 37 ± 2 DEG C, 25 ~ 30rpm revolving speed is mixed 10-14 hours, has been coated with the immune magnetic particle of antibody；Wherein
Antibody additive amount be every 2mg magnetic particle add 10 μ g antibody, the antibody be anti-human epcam antibody, anti-human ICAM-1 antibody,
Anti-human EGFR antibody, anti-human folacin receptor antibody, anti-human CD10 antibody, anti human CD 19 antibody, anti-humen CD 20 antibody or anti-human
CD33 antibody.
The sealer and 100mg/ of 75mg/mL are rapidly added in the centrifuge tube containing the immune magnetic particle for being coated with antibody
Centrifuge tube is put on vertical rotary mixer by the BSA solution of mL after mixing on eddy mixer, in 37 ± 2 DEG C, 25 ~
30rpm revolving speed is closed 2 hours；Centrifuge tube is removed, cleaning solution is added and is resuspended, inhales and abandons after stratification on magnetic frame
Clearly, after being washed repeatedly 2-4 times with cleaning solution, preservation liquid is added and is resuspended, is inhaled after stratification on magnetic frame and abandons supernatant, with guarantor
Liquid storage washes repeatedly 2 times, finally with the immune magnetic particle of liquid resuspension is saved, obtains the immune magnetic particle solution of soilless sticking；It is wherein each
Ingredient single additive amount is that every 2mg magnetic particle adds 200 μ L sealers, 200 μ LBSA solution, 1mL cleaning solution and 1mL preservation liquid,
The sealer is the glycine solution of 75mg/mL, and the cleaning solution is to lead to containing 0.1% Qula and 0.53% polysorbas20
0.9wt% sodium chloride solution, the preservation liquid are the HEPS liquid containing 1.6wt%BSA.
The immune magnetic particle capture instrument is that immune magnetic particle captures instrument CellRichTM, program is set are as follows: sample size is
1.0-7.5mL, acquisition speed 1.0-5.0mL/h, flushing speed are 2.0-7.0mL/h.
The primary antibody of the PD-L1 immunohistochemical staining reagent is that the anti human PD-L 1 of model MAB1561 or ab205921 are anti-
Body, secondary antibody are horseradish peroxidase-labeled mountain sheep anti-mouse igg or horseradish peroxidase-labeled goat anti-rabbit igg.
The primary antibody concentration of the PD-L1 immunohistochemical staining reagent is 0.5mg/ml, secondary antibody concentration is 0.8mg/ml, closing
Liquid is the TBST buffer containing 5vt% lowlenthal serum and developing solution is DAB color developing agent.
2, a kind of special agent detected for cell PD-L1 protein expression in body fluid sample, the reagent include exempting from
Epidemic disease magnetic particle mixed liquor, the immune magnetic particle mixed liquor are that magnetic particle is immunized in coating anti-human epcam, anti-human ICAM-1 is immunized
Magnetic particle is immunized in magnetic particle, anti-human EGFR antibody mediated immunity magnetic particle, anti-human folacin receptor, magnetic particle is immunized in anti-human CD10, anti-human
Magnetic particle is immunized in CD19, magnetic particle is immunized in anti-humen CD 20 and at least one of magnetic particle is immunized in anti-human CD33.
3, a kind of application of the special agent detected for cell PD-L1 protein expression in body fluid sample, described is immune
Application of the magnetic particle mixed liquor in terms of preparing patients with lung cancer CTCs capturing agent, wherein anti-human in immune magnetic particle mixed liquor
Magnetic particle is immunized in EpCAM, anti-human ICAM-1 is immunized magnetic particle, anti-human EGFR antibody mediated immunity magnetic particle and anti-human folacin receptor and is immunized
The mixing mass ratio of magnetic particle is 2:2:2:4；
Application of the immune magnetic particle mixed liquor in terms of preparing patients with lung cancer lung cancer Pleural effusions CTCs capturing agent, wherein exempting from
It is micro- that the immune magnetic particle of magnetic particle, anti-human ICAM-1, anti-human EGFR antibody mediated immunity magnetic is immunized in anti-human epcam in epidemic disease magnetic particle mixed liquor
The mixing mass ratio that magnetic particle is immunized in grain and anti-human folacin receptor is 4:4:1:1；
Application of the immune magnetic particle mixed liquor in terms of preparing leukaemia juvenile cell capturing agent, wherein immune magnetic particle
Magnetic particle is immunized in anti-human CD10 in mixed liquor, magnetic particle is immunized in anti human CD 19, magnetic particle is immunized in anti-humen CD 20 and anti-human CD33 exempts from
The mixing mass ratio of epidemic disease magnetic particle is 1:1:4:4.
4, a kind of special agent detected for cell PD-L1 protein expression in body fluid sample, the reagent includes PD-
L1 immunohistochemical staining reagent, the primary antibody of the PD-L1 immunohistochemical staining reagent are model MAB1561 or ab205921
Anti human PD-L 1 antibody, secondary antibody are horseradish peroxidase-labeled mountain sheep anti-mouse igg or horseradish peroxidase-labeled goat antirabbit
IgG。
Compared with the prior art, the advantages of the present invention are as follows: cell PD-L1 albumen in a kind of detection body fluid sample of the present invention
The detection method of expression, the tumour cell not only include tumour cell or lymphoma cell in blood, further include Pleural effusions
Or the tumour cell of derived from bone marrow；The detection method includes catching for capturing the immune magnetic particle of tumour cell in body fluid sample
Obtain instrument and matched reagent, and the reagent for tumour cell PD-L1 immunohistochemical staining.Present invention is mainly used for 1. different body fluid
The detection of sample (Pleural effusions, marrow and peripheral blood etc.)；2. the immune suppressant drugs such as nivolumab, pembrolizumab
With diagnosis and curative effect monitoring.
Cell PD-L1 method of protein detection in a kind of body fluid sample of the invention selects different types of magnetic particle to be coupled
Different antibody forms specifically immune magnetic particle.Using specific immune magnetic particle, and by immune magnetic particle capture instrument and
Standard matched reagent carries out automatic capture to the cell in different body fluid samples, and the cell captured can use PD-L1 immunohistochemistry
Reagent carries out PD-L1 Protein Detection, has many advantages, such as the easy interpretation of easy to operate, high sensitivity, testing result.
Detailed description of the invention
Fig. 1 is that embodiment 1 carries out immunohistochemical staining result to the tumour cell to fall off in patients with lung adenocarcinoma peripheral blood；
Fig. 2 is that embodiment 2 carries out immunohistochemical staining result to the tumour cell that patients with lung adenocarcinoma Pleural effusions fall off；
Fig. 3 is that embodiment 3 carries out immunohistochemical staining to acute monocytic leukemia bone marrow mononuclear cells.
Specific embodiment
The present invention will be described in further detail below with reference to the embodiments of the drawings.
Embodiment 1
By taking adenocarcinoma of lung blood of cancer patients sample as an example, caught using the immune magnetic particle of Ningbo Mei Jing medical technology Co., Ltd
Obtain instrument CellRichTMAnd its matched reagent captures tumour cell.And it is carried out using detection method subsequent
Detection, the specific steps are as follows:
1, magnetic particle is immunized to prepare and (illustrate with the magnetic particle of 2mg)
1) round (or ellipse) magnetic particle of brown that partial size is 200nm, carboxyl-content 200- the pretreatment of magnetic particle: are selected
400μmol/g；1mL 0.05mol/L MES buffer (acidic buffer) is added in the centrifuge tube containing 2mg magnetic particle,
3 minutes are stood on magnetic frame, inhales and abandons supernatant, 1mL 0.05mol/L MES buffer is added into centrifuge tube, is ultrasonically treated
Afterwards, 3 minutes are stood on magnetic frame, inhales and abandons supernatant, repeatedly 2-4 times altogether；0.5mL 0.05mol/L is added again in centrifuge tube
MES buffer, continuation are ultrasonically treated in Ultrasound Instrument, obtain in the same size, and shape is complete and the magnetic that is evenly distributed is micro-
Grain suspension；
2) magnetic particle activates: being separately added into 100 μ L's in the centrifuge tube of the magnetic particle suspension obtained containing step (1)
The 50mg/mL EDAC solution of 50mg/mL NHS solution (n-hydroxysuccinimide, ready-to-use) activator and 100 μ L
(carbodiimide, ready-to-use) activator, above-mentioned priming reaction liquid is placed on adjustable rotary mixer, in 25 ~ 40rpm,
After 37 ± 2 DEG C of reaction 30min, 1mL HEPS buffer is added and carries out rinse, is washed repeatedly 3 times on magnetic frame, inhales abandoning supernatant and obtain
Magnetic particle after to activation；
3) magnetic particle coupled antibody: taking the centrifuge tube of magnetic particle after four activation obtained containing step (2), corresponding that 10 μ g are added
Anti-human epcam, the anti-human ICAM-1 of 10 μ g, the anti-human EGFR antibody of 10 μ g and 10 μ g anti-human folacin receptor, by centrifuge tube
Be put into after sealing on vertical rotary mixer, in 37 DEG C, 25 ~ 30rpm revolving speed is mixed 12 hours, respectively obtain be coated with it is anti-human
Magnetic particle is immunized in EpCAM, anti-human ICAM-1 is immunized magnetic particle, anti-human EGFR antibody mediated immunity magnetic particle and anti-human folacin receptor and is immunized
Magnetic particle；
4) it closes: weighing a certain amount of glycine, use H2O dissolves the sealer for being configured to 75mg/mL；It is respectively drawn with liquid-transfering gun
The BSA solution of 200 μ L sealers and 100mg/mL are rapidly added in the centrifuge tube of the immune magnetic particle containing coupled antibody and seal,
After being mixed on eddy mixer, centrifuge tube is put on vertical rotary mixer, in 37 DEG C, the closing 2 of 25 ~ 30rpm revolving speed is small
When；
5) it washs: centrifuge tube is removed, 1mL cleaning solution is added and (leads to containing 0.1% Qula, the 0.9wt% sodium chloride of 0.53% polysorbas20
Solution) be resuspended, stood on magnetic frame suction in 3 minutes abandon supernatant, with cleaning solution wash repeatedly 3 times after, be added 1mL save liquid into
Row is resuspended, and suction in 3 minutes is stood on magnetic frame and abandons supernatant, with liquid (the HEPS liquid containing 1.6wt%BSA) repeated washing is saved 2 times, most
Liquid is saved with 1mL afterwards, immune magnetic particle is resuspended, obtain the immune magnetic particle solution of soilless sticking；Being coated with after washing is anti-human
Magnetic particle is immunized in EpCAM, anti-human ICAM-1 is immunized magnetic particle, anti-human EGFR antibody mediated immunity magnetic particle and anti-human folacin receptor and is immunized
The magnetic particle ratio of 2:2:2:4 in mass ratio is mixed, and is finally obtained mixed for the immune magnetic particle of patients with lung cancer CTC separation
Closing liquid, (2mg/mL, the concentration are the amount of 4 kinds of immune magnetic particles in the solution, are not a kind of independent immune magnetic particle in solution
In amount).
2, the capture of lung cancer CTCs
1) blood sample pre-processes: after blood sample gentle inversion is mixed 8 times, 5mL blood sample (containing anti-coagulants) is drawn into 15mL centrifuge tube,
And suitable Sample dilution (the PBS liquid containing 5wt%BSA) is supplemented, slight reverse, mixing is for several times；By its 800g at room temperature,
Supernatant is removed after centrifugation 10min, until at from precipitating 1mL；
2) it is incubated for: 3mL Sample dilution being added into above-mentioned 15mL centrifuge tube and magnetic particle mixed liquor is immunized in 200 μ L, it is slight to run
It mixes for several times；Centrifuge tube is fixed on vertical mixed instrument, 1.5h is incubated at 37 DEG C with the speed of 8-10rpm, makes CTCs
It is combined with immune magnetic particle；
3) it captures CTCs cell: the centrifuge tube of liquid will be resuspended, be placed in immune magnetic particle capture instrument CellRichTM, carry out
The separation and capture of CTCs cell.Program is set are as follows: sample size 4.5mL, acquisition speed 2.5mL/h, rinsing speed is
5.0mL/h captures careful taking-up glass slide after the completion of program.
3, the PD-L1 Protein Detection of lung cancer CTCs
1) fixed closed: acetone (covering slide) is added dropwise on the Cell sheet glass of capture, 4 DEG C of fixed 20min, room temperature is dry after abandoning liquid
Dry 5min；PBS buffer solution rinses 3 times, each 2min；3wt% hydrogen peroxide is added dropwise and stands 15min so that Endogenous peroxide
Then enzyme-deactivating is rinsed 3 times, each 2min with PBS buffer solution；Confining liquid (the TBST buffering containing 5vt% lowlenthal serum is added dropwise
Liquid), 37 DEG C of incubation 20min abandon liquid；
2) it is incubated for antibody: primary antibody being added on Cell sheet glass and covers cell, 4 DEG C of overnight incubations abandon liquid；PBST buffer rinsing 3
It is secondary, each 5min；ELIAS secondary antibody is added and covers tissue, 37 DEG C of incubation 30min abandon liquid；PBST buffer rinsing 3 times, every time
5min；Wherein primary antibody is the anti human PD-L 1 antibody of the model MAB1561 or ab205921 of concentration 0.5mg/mL, and secondary antibody is dense
Spend the horseradish peroxidase-labeled mountain sheep anti-mouse igg or horseradish peroxidase-labeled goat anti-rabbit igg of 0.8mg/mL；
3) colour developing and mounting: being added DAB color developing agent in Cell sheet glass, be stored at room temperature 5min, in microscopic observation colour developing situation；With
dH2After O is rinsed, reuses 0.01M PBS buffer solution and carry out rinsing 2min, redyed using haematoxylin: haematoxylin dyeing
The aobvious indigo plant of 2min, 40 DEG C of warm water 20s, is dehydrated transparent, mounting microscopy.
Interpretation of result: selection partial size is 200nm, and the magnetic particle of 200-400 μm of ol/g of carboxyl-content is with higher to compare table
The advantages that area, magnetic responsiveness and lower toxicity.It being centrifuged through overactivation, magnetic particle can be evenly dispersed in the solution, thus, it is possible to
Efficient coupled antibody (the efficient coupled antibody of carboxyl energy of magnetic particle pan coating).Magnetic particle is immunized using this, and (coupling is anti-human
EpCAM, anti-human ICAM-1, anti-human EGFR antibody and anti-human folacin receptor) capture cell when, because its high magnetic responsiveness can make to capture
Speed gets a promotion, and acquisition procedure can be completed in 1.5h, shortens 15min than previous capture time.While speed-raising, because
Human peripheral blood has carried out the pretreatments such as washing centrifugation, removes partial impurities, so that capture rate be made to be improved.By the thin of capture
Born of the same parents carry out PD-L1 immunohistochemical staining, and for experimental result as shown in Figure 1, remaining the understain of lymphocyte after birth in cell sheet, lung cancer is thin
Born of the same parents' (heteromorphic nucleus) are less, and cell membrane is in light brown, and PD-L1 expresses weakly positive.
Embodiment 2
By taking adenocarcinoma of lung tumor patient chest and abdomen water sample as an example, detection method detects it.
1, magnetic particle is immunized to prepare and (illustrate with the magnetic particle of 2mg)
1) round (or ellipse) magnetic particle of brown that partial size is 200nm, carboxyl-content 200- the pretreatment of magnetic particle: are selected
400μmol/g；1mL 0.05mol/L MES buffer (acidic buffer) is added in the centrifuge tube containing 2mg magnetic particle,
3 minutes are stood on magnetic frame, inhales and abandons supernatant, 1mL 0.05mol/L MES buffer is added into centrifuge tube, is ultrasonically treated
Afterwards, 3 minutes are stood on magnetic frame, inhales and abandons supernatant, repeatedly 2-4 times altogether；0.5mL 0.05mol/L is added again in centrifuge tube
MES buffer, continuation are ultrasonically treated in Ultrasound Instrument, obtain in the same size, and shape is complete and the magnetic that is evenly distributed is micro-
Grain suspension；
2) magnetic particle activates: being separately added into 100 μ L's in the centrifuge tube of the magnetic particle suspension obtained containing step (1)
The 50mg/mL EDAC solution of 50mg/mL NHS solution (n-hydroxysuccinimide, ready-to-use) activator and 100 μ L
(carbodiimide, ready-to-use) activator, above-mentioned priming reaction liquid is placed on adjustable rotary mixer, in 25 ~ 40rpm,
After 37 ± 2 DEG C of reaction 30min, 1mL HEPS buffer is added and carries out rinse, is washed repeatedly 3 times on magnetic frame, inhales abandoning supernatant and obtain
Magnetic particle after to activation；
3) magnetic particle coupled antibody: taking the centrifuge tube of magnetic particle after four activation obtained containing step (2), corresponding that 10 μ g are added
Anti-human epcam, the anti-human ICAM-1 of 10 μ g, the anti-human EGFR antibody of 10 μ g and 10 μ g anti-human folacin receptor, by centrifuge tube
Be put into after sealing on vertical rotary mixer, in 37 DEG C, 25 ~ 30rpm revolving speed is mixed 12 hours, respectively obtain be coated with it is anti-human
Magnetic particle is immunized in EpCAM, anti-human ICAM-1 is immunized magnetic particle, anti-human EGFR antibody mediated immunity magnetic particle and anti-human folacin receptor and is immunized
Magnetic particle；
4) it closes: weighing a certain amount of glycine, use H2O dissolves the sealer for being configured to 75mg/mL；It is respectively drawn with liquid-transfering gun
The BSA solution of 200 μ L sealers and 100mg/mL are rapidly added in the centrifuge tube of the immune magnetic particle containing coupled antibody and seal,
After being mixed on eddy mixer, centrifuge tube is put on vertical rotary mixer, in 37 DEG C, the closing 2 of 25 ~ 30rpm revolving speed is small
When；
5) it washs: centrifuge tube is removed, 1mL cleaning solution is added and (leads to containing 0.1% Qula, the 0.9wt% sodium chloride of 0.53% polysorbas20
Solution, since Qula is logical and polysorbas20 is liquid, the two proportional amount refers to volume ratio) it is resuspended, 3 points are stood on magnetic frame
Clock, which is inhaled, abandons supernatant, after being washed repeatedly 3 times with cleaning solution, 1mL preservation liquid is added and is resuspended, the abandoning of suction in 3 minutes is stood on magnetic frame
Supernatant is washed repeatedly 2 times with liquid (the HEPS liquid containing 1.6wt%BSA) is saved, finally saves liquid with 1mL and immune magnetic particle is resuspended,
Obtain the immune magnetic particle solution of soilless sticking；By being coated with after washing, magnetic particle is immunized in anti-human epcam, anti-human ICAM-1 is immunized
Magnetic particle, anti-human EGFR antibody mediated immunity magnetic particle and anti-human folacin receptor be immunized the magnetic particle ratio of 4:4:1:1 in mass ratio into
Row mixing, finally obtains the immune magnetic particle mixed liquor (2mg/mL) for patients with lung cancer CTC separation.
2, the capture of lung cancer Pleural effusions CTCs
1) chest and abdomen water pretreatment: after Pleural effusions gentle inversion is mixed, take the Pleural effusions of 45mL to 50mL centrifuge tube, at room temperature
700g is abandoned supernatant up to the solution of bottom residue 5mL after being centrifuged 5min, is mended using Sample dilution (the PBS liquid containing 5wt%BSA)
It is charged to 45mL, after being mixed by inversion at room temperature, 700g collects the cell of bottom after being centrifuged 5min；
2) hatching combination: additional sample dilution to 5mL, be resuspended cell after go in 15mL centrifuge tube be added 200 μ L be immunized magnetic
Particle mixed liquor is slightly mixed by inversion for several times；Centrifuge tube is fixed on vertical mixed instrument, with the speed of 8-10 rpm at 37 DEG C
1.5 h of lower incubation, are combined CTCs and immune magnetic particle；
3) it captures CTCs: the centrifuge tube of liquid will be resuspended, be placed in immune magnetic particle capture instrument CellRichTM, carry out CTCs
Capture with separate.Program is set are as follows: sample size is 1 mL, and acquisition speed is 1 mL/h, and flushing speed is 2.0 mL/h, capture
Careful taking-up glass slide after the completion of program.
3, the PD-L1 Protein Detection of lung cancer Pleural effusions CTCs
1) fixed closed: acetone (covering slide) is added dropwise on the Cell sheet glass of capture, 4 DEG C of fixed 20min, room temperature is dry after abandoning liquid
Dry 5min；PBS buffer solution rinses 3 times, each 2min；3wt% hydrogen peroxide is added dropwise and stands 15min so that Endogenous peroxide
Then enzyme-deactivating is rinsed 3 times, each 2min with PBS buffer solution；Confining liquid (the TBST buffering containing 5vt% lowlenthal serum is added dropwise
Liquid), 37 DEG C of incubation 20min abandon liquid；
2) it is incubated for antibody: primary antibody being added on Cell sheet glass and covers cell, 4 DEG C of overnight incubations abandon liquid；PBST buffer rinsing 3
It is secondary, each 5min；ELIAS secondary antibody is added and covers tissue, 37 DEG C of incubation 30min abandon liquid；PBST buffer rinsing 3 times, every time
5min；Wherein primary antibody is the anti human PD-L 1 antibody of the model MAB1561 or ab205921 of concentration 0.5mg/mL, and secondary antibody is dense
Spend the horseradish peroxidase-labeled mountain sheep anti-mouse igg or horseradish peroxidase-labeled goat anti-rabbit igg of 0.8mg/mL；
3) colour developing and mounting: being added DAB color developing agent in Cell sheet glass, be stored at room temperature 5min, in microscopic observation colour developing situation；With
dH2After O is rinsed, reuses 0.01M PBS buffer solution and carry out rinsing 2min, redyed using haematoxylin: haematoxylin dyeing
The aobvious indigo plant of 2min, 40 DEG C of warm water 20s, is dehydrated transparent, mounting microscopy.
Interpretation of result: being 200nm with partial size, and the magnetic particle homogeneity of 200-400 μm of ol/g of carboxyl-content is good, and granularity is suitable
In.Partial size is too small, and there are dipole-dipole effects for intergranular, so that being easy to reunite between particle.Magnetic particle is centrifuged through overactivation, coupling
After antibody, can good dispersion in the solution, do not reunite.It can remove impurity after Pleural effusions are carried out washing centrifugation, use coating
The immune magnetic particle (coupling anti-human epcam, anti-human ICAM-1, anti-human EGFR antibody and anti-human folacin receptor) of antibody is caught
It obtains, quickly can closely combine CTCs, the precision of capture is thus promoted to 86%.Experimental result is as shown in Fig. 2, cell sheet
Middle lung carcinoma cell (heteromorphic nucleus) cell membrane is in brown, and PD-L1 expression is positive.
Embodiment 3
The capture of Bone Marrow of Patients leukaemia cell is enriched with: Bone Marrow of Patients sample being carried out density gradient centrifugation first and is collected in sample
Mononuclearcell, with pretreatment fluid to the immune magnetic particle capture instrument CellRich collected after cell is resuspended with our companyTM
And its matched reagent carries out the capture of leukaemia juvenile cell.
1, magnetic particle is immunized to prepare and (illustrate with the magnetic particle of 2mg)
1) pretreatment of magnetic particle: the partial size of the magnetic particle of selection is 1 μm, 200-600 μm of ol/g of carboxyl-content, and shape is circle
Shape, appearance are brown；1mL 0.05mol/L MES buffer (acidic buffer is added in the centrifuge tube containing 2mg magnetic particle
Liquid), 3 minutes are stood on magnetic frame, inhales and abandons supernatant, 1mL 0.05mol/L MES buffer is added into centrifuge tube, is surpassed
After sonication, 3 minutes are stood on magnetic frame, inhales and abandons supernatant, repeatedly 2-4 times altogether；0.5mL is added again in centrifuge tube
0.05mol/L MES buffer, continuation are ultrasonically treated in Ultrasound Instrument, obtain in the same size, and shape is complete and distribution
Uniform magnetic particle suspension；
2) magnetic particle activates: being separately added into 100 μ L's in the centrifuge tube of the magnetic particle suspension obtained containing step (1)
The 50mg/mL EDAC solution of 50mg/mL NHS solution (n-hydroxysuccinimide, ready-to-use) activator and 100 μ L
(carbodiimide, ready-to-use) activator, above-mentioned priming reaction liquid is placed on adjustable rotary mixer, in 25 ~ 40rpm,
After 37 ± 2 DEG C of reaction 30min, 1mL HEPS buffer is added and carries out rinse, is washed repeatedly 3 times on magnetic frame, inhales abandoning supernatant and obtain
Magnetic particle after to activation；
3) magnetic particle coupled antibody: taking the centrifuge tube of magnetic particle after four activation obtained containing step (2), corresponding that 10 μ g are added
Anti-human CD10, the anti human CD 19 of 10 μ g, the anti-humen CD 20 of 10 μ g and 10 μ g anti-human CD33, will centrifugation the seal of tube after be put into it is vertical
On straight impeller, in 37 DEG C, 25 ~ 30rpm revolving speed is mixed 12 hours, is respectively obtained and has been coated with anti-human CD10 that magnetic is immunized is micro-
Magnetic particle is immunized in grain, anti human CD 19, magnetic particle is immunized in anti-humen CD 20 and magnetic particle is immunized in anti-human CD33；
4) it closes: weighing a certain amount of glycine, use H2O dissolves the sealer for being configured to 75mg/mL；It is respectively drawn with liquid-transfering gun
The BSA solution of 200 μ L sealers and 200 μ L 100mg/mL are rapidly added the centrifuge tube of the immune magnetic particle containing coupled antibody
Centrifuge tube is put on vertical rotary mixer by middle sealing after mixing on eddy mixer, in 37 DEG C, 25 ~ 30rpm revolving speed
Closing 2 hours；
5) it washs: centrifuge tube is removed, 1mL cleaning solution is added and (leads to containing 0.1% Qula, the 0.9wt% sodium chloride of 0.53% polysorbas20
Solution) be resuspended, stood on magnetic frame suction in 3 minutes abandon supernatant, with cleaning solution wash repeatedly 3 times after, be added 1mL save liquid into
Row is resuspended, and suction in 3 minutes is stood on magnetic frame and abandons supernatant, with liquid (the HEPS liquid containing 1.6wt%BSA) repeated washing is saved 2 times, most
Liquid is saved with 1mL afterwards, immune magnetic particle is resuspended, obtain the immune magnetic particle solution of soilless sticking；Being coated with after washing is anti-human
Magnetic particle is immunized in CD10, magnetic particle is immunized in anti human CD 19, magnetic particle is immunized in anti-humen CD 20 and magnetic particle is immunized by matter in anti-human CD33
It measures the ratio than 1:1:4:4 to be mixed, finally obtains the immune magnetic particle mixed liquor (2mg/mL) for capturing juvenile cell.
2, the capture of leukaemia juvenile cell
1) it sample preprocessing: takes and punctures resulting Bone Marrow of Patients, be slowly injected into laggard line density gradient centrifugation in centrifuge tube；It draws
Tunica albuginea confluent monolayer cells are washed cell 2 times using PBS buffer solution, then with 3mL pretreatment fluid (the PBS liquid containing 5wt%BSA) to cell
It is resuspended；
2) hatching combination: the cell of resuspension is transferred to the immune magnetic particle mixed liquor that 500 μ L are added in 15mL centrifuge tube, slightly
It is mixed by inversion for several times；Centrifuge tube is fixed on vertical mixed instrument, 1.5h is incubated at 37 DEG C with the speed of 8-10rpm, makes children
Naive cell and immune magnetic particle are combined；
3) capture of juvenile cell: will be resuspended the centrifuge tube of liquid, be placed in immune magnetic particle capture instrument CellRichTM, into
The capture of row cell with separate.Program is set are as follows: sample size 7.5mL, acquisition speed 5.0mL/h, rinsing speed is
7.0mL/h captures careful taking-up glass slide after the completion of program.
3, the PD-L1 Protein Detection of juvenile cell
1) fixed closed: acetone (covering slide) is added dropwise on the Cell sheet glass of capture, 4 DEG C of fixed 20min, room temperature is dry after abandoning liquid
Dry 5min；PBS buffer solution rinses 3 times, each 2min；3wt% hydrogen peroxide is added dropwise and stands 15min so that Endogenous peroxide
Then enzyme-deactivating is rinsed 3 times, each 2min with PBS buffer solution；Confining liquid (the TBST buffering containing 5vt% lowlenthal serum is added dropwise
Liquid), 37 DEG C of incubation 20min abandon liquid；
2) it is incubated for antibody: primary antibody being added on Cell sheet glass and covers cell, 4 DEG C of overnight incubations abandon liquid；PBST buffer rinsing 3
It is secondary, each 5min；ELIAS secondary antibody is added and covers tissue, 37 DEG C of incubation 30min abandon liquid；PBST buffer rinsing 3 times, every time
5min；Wherein primary antibody is the anti human PD-L 1 antibody of the model MAB1561 or ab205921 of concentration 0.5mg/mL, and secondary antibody is dense
Spend the horseradish peroxidase-labeled mountain sheep anti-mouse igg or horseradish peroxidase-labeled goat anti-rabbit igg of 0.8mg/mL；
3) colour developing and mounting: being added DAB color developing agent in Cell sheet glass, be stored at room temperature 5min, in microscopic observation colour developing situation；With
dH2After O is rinsed, reuses 0.01M PBS buffer solution and carry out rinsing 2min, redyed using haematoxylin: haematoxylin dyeing
The aobvious indigo plant of 2min, 40 DEG C of warm water 20s, is dehydrated transparent, mounting microscopy.
Interpretation of result: selecting partial size is 1 μm, and shape is circular magnetic particle, compared with the magnetic particle of other shapes, tool
There is bigger surface area, can capture in conjunction with more antibody.Magnetic particle suspension by washing, activation, centrifugation and etc., removal
Shape is incomplete, and the excessive particle of partial size improves the joint efficiency (78%) of magnetic particle and antibody, while also saving cost.
Can be evenly dispersed in the solution after magnetic particle coupled antibody (anti-human CD10, anti human CD 19, anti-humen CD 20, anti-human CD33), it reduces
Agglomeration.When magnetic particle capture is immunized using this, because its high magnetic responsiveness can make acquisition speed get a promotion, capture time
Shorten 20min.For experimental result as shown in figure 3, leukaemia juvenile cell after birth contaminates deeply in cell sheet, PD-L1 expression is positive.
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common
Within the essential scope of the present invention, the variations, modifications, additions or substitutions made also should belong to protection of the invention to technical staff
Range.
Claims (10)
1. the detection method of cell PD-L1 protein expression in a kind of body fluid sample, it is characterised in that the following steps are included: will preparation
Immune magnetic particle mixed liquor in conjunction with neoplasm circulating cells in body fluid sample, be subsequently placed in immune magnetic particle capture instrument and carry out
Capture, the tumour cell of capture is dyed by PD-L1 immunohistochemical staining reagent, assesses the protein expression of PD-L1.
2. the detection method of cell PD-L1 protein expression, feature exist in a kind of body fluid sample according to claim 1
In: the immune magnetic particle mixed liquor is that magnetic particle is immunized in coating anti-human epcam, magnetic particle is immunized in anti-human ICAM-1, anti-human
It is micro- that magnetic particle is immunized in EGFR antibody mediated immunity magnetic particle, anti-human folacin receptor, magnetic particle is immunized in anti-human CD10, magnetic is immunized in anti human CD 19
Magnetic particle is immunized in grain, anti-humen CD 20 and at least one of magnetic particle is immunized in anti-human CD33.
3. the detection method of cell PD-L1 protein expression in a kind of body fluid sample according to claim 2, it is characterised in that
Specific step is as follows for the preparation method of the immune magnetic particle:
(1) pretreatment of magnetic particle: 0.05mol/L MES buffer is added in the centrifuge tube containing magnetic particle, by centrifuge tube
It is placed on magnetic frame after stratification, inhales and abandon supernatant, the 0.05mol/L MES buffer of equivalent is added into centrifuge tube, carry out
It after ultrasonic treatment, is placed on magnetic frame after stratification, inhales and abandon supernatant, repeat the above steps 2-4 times；Add again in centrifuge tube
Enter 0.5mL 0.05mol/L MES buffer, continuation is ultrasonically treated in Ultrasound Instrument, obtains that in the same size, shape is complete
And the magnetic particle suspension being evenly distributed；Wherein additive amount of MES buffer is that every 2mg magnetic particle addition 1mL MES is slow
Fliud flushing；Magnetic particle is the brown round or ellipse magnetic particle of partial size 200-1000nm, and carboxyl-content is 200 μm of ol/g-600 μ
mol/g；
(2) magnetic particle activates: 50mg/mL NHS solution activator and 50mg/mL EDAC are separately added into magnetic particle suspension
Solution activator is subsequently placed on adjustable rotary mixer, and after 25 ~ 40rpm, 37 ± 2 DEG C of reaction 30min, HEPS is added
Buffer carries out rinse, is placed on magnetic frame and washes repeatedly 2-4 times, inhales and abandons magnetic particle after supernatant is activated；Wherein NHS is molten
The additive amount of liquid, EDAC solution and HEPS buffer be every 2mg magnetic particle add 100 μ LNHS solution, 100 μ LEDAC solution and
1mL HEPS buffer；
(3) magnetic particle coupled antibody: after antibody sealing is added in the centrifuge tube of magnetic particle after containing activation, it is put into vertical rotary
On mixer, in 37 ± 2 DEG C, 25 ~ 30rpm revolving speed is mixed 10-14 hours, has been coated with the immune magnetic particle of antibody；Wherein
Antibody additive amount be every 2mg magnetic particle add 10 μ g antibody, the antibody be anti-human epcam antibody, anti-human ICAM-1 antibody,
Anti-human EGFR antibody, anti-human folacin receptor antibody, anti-human CD10 antibody, anti human CD 19 antibody, anti-humen CD 20 antibody or anti-human
CD33 antibody.
4. the detection method of cell PD-L1 protein expression, feature exist in a kind of body fluid sample according to claim 3
In: it is rapidly added in the centrifuge tube containing the immune magnetic particle that has been coated with antibody the sealer and 100mg/mL of 75mg/mL
Centrifuge tube is put on vertical rotary mixer by BSA solution after mixing on eddy mixer, in 37 ± 2 DEG C, 25 ~ 30rpm
Revolving speed is closed 2 hours；Centrifuge tube is removed, cleaning solution is added and is resuspended, is inhaled after stratification on magnetic frame and abandons supernatant, with clear
After washing lotion repeated washing 2-4 times, preservation liquid is added and is resuspended, is inhaled after stratification on magnetic frame and abandons supernatant, with preservation liquid weight
After backwashing is washed 2 times, finally with the immune magnetic particle of liquid resuspension is saved, obtains the immune magnetic particle solution of soilless sticking；Wherein each ingredient list
Secondary additive amount is that every 2mg magnetic particle adds 200 μ L sealers, 200 μ LBSA solution, 1mL cleaning solution and 1mL preservation liquid, described
Sealer is the glycine solution of 75mg/mL, and the cleaning solution is containing the logical 0.9wt% with 0.53% polysorbas20 of 0.1% Qula
Sodium chloride solution, the preservation liquid are the HEPS liquid containing 1.6wt%BSA.
5. the detection method of cell PD-L1 protein expression, feature exist in a kind of body fluid sample according to claim 1
In: the immune magnetic particle capture instrument is that immune magnetic particle captures instrument CellRichTM, program is set are as follows: sample size 1.0-
7.5mL, acquisition speed 1.0-5.0mL/h, flushing speed are 2.0-7.0mL/h.
6. the detection method of cell PD-L1 protein expression, feature exist in a kind of body fluid sample according to claim 1
In the anti human PD-L 1 antibody that: the primary antibody of the PD-L1 immunohistochemical staining reagent is model MAB1561 or ab205921, two
Resist for horseradish peroxidase-labeled mountain sheep anti-mouse igg or horseradish peroxidase-labeled goat anti-rabbit igg.
7. the detection method of cell PD-L1 protein expression, feature exist in a kind of body fluid sample according to claim 6
In: the primary antibody concentration of the PD-L1 immunohistochemical staining reagent is 0.5mg/ml, secondary antibody concentration is 0.8mg/ml, confining liquid is
TBST buffer and developing solution containing 5vt% lowlenthal serum are DAB color developing agent.
8. a kind of special agent detected for cell PD-L1 protein expression in body fluid sample described in claim 1, feature
Be: the reagent includes immune magnetic particle mixed liquor, and the immune magnetic particle mixed liquor is that coating anti-human epcam is immune
Magnetic particle, anti-human ICAM-1 are immunized magnetic particle, anti-human EGFR antibody mediated immunity magnetic particle, anti-human folacin receptor and magnetic particle are immunized, resists
Magnetic particle is immunized in people CD10, magnetic particle is immunized in anti human CD 19, magnetic particle is immunized in anti-humen CD 20 and anti-human CD33 is immunized in magnetic particle
At least one.
9. a kind of application according to any one of claims 8 for the special agent of cell PD-L1 protein expression detection in body fluid sample,
It is characterized by: application of the immune magnetic particle mixed liquor in terms of preparing patients with lung cancer CTCs capturing agent, wherein being immunized
Magnetic particle is immunized in anti-human epcam in magnetic particle mixed liquor, magnetic particle, anti-human EGFR antibody mediated immunity magnetic particle is immunized in anti-human ICAM-1
The mixing mass ratio that magnetic particle is immunized with anti-human folacin receptor is 2:2:2:4；
Application of the immune magnetic particle mixed liquor in terms of preparing patients with lung cancer lung cancer Pleural effusions CTCs capturing agent, wherein exempting from
It is micro- that the immune magnetic particle of magnetic particle, anti-human ICAM-1, anti-human EGFR antibody mediated immunity magnetic is immunized in anti-human epcam in epidemic disease magnetic particle mixed liquor
The mixing mass ratio that magnetic particle is immunized in grain and anti-human folacin receptor is 4:4:1:1；
Application of the immune magnetic particle mixed liquor in terms of preparing leukaemia juvenile cell capturing agent, wherein immune magnetic particle
Magnetic particle is immunized in anti-human CD10 in mixed liquor, magnetic particle is immunized in anti human CD 19, magnetic particle is immunized in anti-humen CD 20 and anti-human CD33 exempts from
The mixing mass ratio of epidemic disease magnetic particle is 1:1:4:4.
10. a kind of special agent detected for cell PD-L1 protein expression in body fluid sample described in claim 1, special
Sign is: the reagent includes PD-L1 immunohistochemical staining reagent, the primary antibody of the PD-L1 immunohistochemical staining reagent
For the anti human PD-L 1 antibody of model MAB1561 or ab205921, secondary antibody be horseradish peroxidase-labeled mountain sheep anti-mouse igg or
Horseradish peroxidase-labeled goat anti-rabbit igg.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111307789A (en) * | 2019-12-18 | 2020-06-19 | 郑州安图生物工程股份有限公司 | Folic acid detection kit and detection method |
| CN111537726A (en) * | 2020-05-29 | 2020-08-14 | 武汉大学 | Method for efficiently and quantitatively detecting PD-L1 level in extracellular vesicle, ELISA kit and using method |
| CN112730839A (en) * | 2021-01-20 | 2021-04-30 | 宁波海壹生物科技有限公司 | Kit for measuring content of cytokeratin 19 fragments by magnetic particle chemiluminescence method |
| CN114636820A (en) * | 2022-05-07 | 2022-06-17 | 珠海圣美生物诊断技术有限公司 | Kit and method for detecting circulating PD-L1 positive cells |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102405411A (en) * | 2009-03-18 | 2012-04-04 | 加利福尼亚大学董事会 | Device for capturing circulating cells |
| CN103630440A (en) * | 2013-11-28 | 2014-03-12 | 武汉大学 | Enriching method of circulating tumor cells |
| US20140141986A1 (en) * | 2011-02-22 | 2014-05-22 | David Spetzler | Circulating biomarkers |
| CN104178454A (en) * | 2013-05-24 | 2014-12-03 | 益善生物技术股份有限公司 | Enrichment and analysis method for circulating tumor cells |
| CN105717298A (en) * | 2016-01-29 | 2016-06-29 | 浙江数问生物技术有限公司 | PD-L1 immunohistochemical kit |
| CN106461668A (en) * | 2014-03-07 | 2017-02-22 | 伊利诺伊大学评议会 | Biomimetic microfluid device for capturing circulating tumor cells |
| CN106939281A (en) * | 2017-03-14 | 2017-07-11 | 复旦大学附属中山医院 | Circulating tumor cell sorter and its kit |
| CN106978400A (en) * | 2016-12-13 | 2017-07-25 | 无锡傲锐东源生物科技有限公司 | Anti- PD L1 protein monoclonal antibodies and application thereof |
| CN108179134A (en) * | 2017-12-27 | 2018-06-19 | 武汉大学 | Based on EpCAM/PSMA double antibody functionalization micro-flow control chips and its preparation method and application |
| CN108186603A (en) * | 2018-02-08 | 2018-06-22 | 上海菱蓝生物科技有限公司 | A kind of silica nano material delivery system for capturing and treating for circulating tumor cell |
-
2019
- 2019-03-04 CN CN201910159426.4A patent/CN109975554A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102405411A (en) * | 2009-03-18 | 2012-04-04 | 加利福尼亚大学董事会 | Device for capturing circulating cells |
| US20140141986A1 (en) * | 2011-02-22 | 2014-05-22 | David Spetzler | Circulating biomarkers |
| CN104178454A (en) * | 2013-05-24 | 2014-12-03 | 益善生物技术股份有限公司 | Enrichment and analysis method for circulating tumor cells |
| CN103630440A (en) * | 2013-11-28 | 2014-03-12 | 武汉大学 | Enriching method of circulating tumor cells |
| CN106461668A (en) * | 2014-03-07 | 2017-02-22 | 伊利诺伊大学评议会 | Biomimetic microfluid device for capturing circulating tumor cells |
| CN105717298A (en) * | 2016-01-29 | 2016-06-29 | 浙江数问生物技术有限公司 | PD-L1 immunohistochemical kit |
| CN106978400A (en) * | 2016-12-13 | 2017-07-25 | 无锡傲锐东源生物科技有限公司 | Anti- PD L1 protein monoclonal antibodies and application thereof |
| CN106939281A (en) * | 2017-03-14 | 2017-07-11 | 复旦大学附属中山医院 | Circulating tumor cell sorter and its kit |
| CN108179134A (en) * | 2017-12-27 | 2018-06-19 | 武汉大学 | Based on EpCAM/PSMA double antibody functionalization micro-flow control chips and its preparation method and application |
| CN108186603A (en) * | 2018-02-08 | 2018-06-22 | 上海菱蓝生物科技有限公司 | A kind of silica nano material delivery system for capturing and treating for circulating tumor cell |
Non-Patent Citations (1)
| Title |
|---|
| 陆松梅 等: "EPCAM、MUC16免疫磁珠制备及循环肿瘤细胞检测", 《EPCAM、MUC16免疫磁珠制备及循环肿瘤细胞检测》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111307789A (en) * | 2019-12-18 | 2020-06-19 | 郑州安图生物工程股份有限公司 | Folic acid detection kit and detection method |
| CN111307789B (en) * | 2019-12-18 | 2023-06-06 | 郑州安图生物工程股份有限公司 | Folic acid detection kit and detection method |
| CN111537726A (en) * | 2020-05-29 | 2020-08-14 | 武汉大学 | Method for efficiently and quantitatively detecting PD-L1 level in extracellular vesicle, ELISA kit and using method |
| CN112730839A (en) * | 2021-01-20 | 2021-04-30 | 宁波海壹生物科技有限公司 | Kit for measuring content of cytokeratin 19 fragments by magnetic particle chemiluminescence method |
| CN112730839B (en) * | 2021-01-20 | 2024-01-30 | 宁波海尔施智造有限公司 | Kit for measuring content of cytokeratin 19 fragments by magnetic particle chemiluminescence method |
| CN114636820A (en) * | 2022-05-07 | 2022-06-17 | 珠海圣美生物诊断技术有限公司 | Kit and method for detecting circulating PD-L1 positive cells |
| CN114636820B (en) * | 2022-05-07 | 2022-10-21 | 珠海圣美生物诊断技术有限公司 | Kit and method for detecting circulating PD-L1 positive cells |
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