CN107290537A - Detect the flow cytometer detection kit of tumor markers - Google Patents
Detect the flow cytometer detection kit of tumor markers Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
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Abstract
The present invention relates to field of biological medicine, in particular to a kind of flow cytometer detection kit for detecting tumor markers, including to mark the CD14 antibody and CD16 antibody of Cell membrane antigens, and to mark the antibody of endochylema internal antigens；The endochylema internal antigens are TKTL1 and/or DNASE1L1.
Description
Technical field
The present invention relates to field of biological medicine, in particular to a kind of flow cytometer detection for detecting tumor markers
Kit.
Background technology
Monocytes/macrophages include huge in the premonocyte in marrow, the monocyte in peripheral blood and tissue
Phagocyte.Human peripheral's haemocyte is broadly divided into cytode (predominantly erythrocyte) and karyocyte (leucocyte etc.).It is single
Nucleus accounts for 5% or so of karyocyte sum, is antigen submission in immune system by medullary system hematopoietic progenitor cell
The important component of cell (APC cells).
In human peripheral blood circulation, leucocyte is distributed in two places, i.e. circulatory pool and storage pool, and circulatory pool is exactly
Leucocyte in fluid flow blood, storage pool is just attached to vascular wall, lymph node, the leucocyte of marrow etc., is temporarily not involved in
Leucocyte in leucocyte and circulatory pool in blood circulation, but storage pool keeps dynamic equilibrium.Leucocyte is in human circulation system
It is ripe in system to break up and realize that immunologic function be roughly divided into following steps：Monocyte in capillary → pass through blood
Tissue outside tube wall intravasation → discovery heterologous material (bacterium, pathogen, tumour cell etc.) → is divided into the macrophage of maturation
Cell → phagocytosis and digest → by antigen submission to specific cell surface molecule → via lymphatic vessel be back to peripheral blood →
Complete antigen submission and activate lethal immunocyte.
As the external pathogen invasion of fight and the sentry for understanding itself abnormal cell mechanism, macrophage is immune
The important step of system formation immune response, is acted on great.Have all the time in normal human towards anti-tumor (immortality
Change, improper apoptosis) cell of development produces, normal immunity of organism mechanism can be removed it in time by said process.
As can be seen here no matter in healthy population or cancer patient's body macrophage for anti-tumor cell phagocytosis, when being all no
Without carving not in progress, for the observation of tumour source material in monocytes/macrophages (including protein, accounting, lipid etc.),
The new perspective that a monitoring in-vivo tumour occurs, developed can be provided for us, its application prospect is inevitable widely.
Monocytes/macrophages are as the important component of karyocyte, and laboratory is generally by its surface antigen molecules
What CD14 and CD16 were detected, it is currently available the antibody of maturation.Main clinical practice concentrates on haemocyte composition inspection
Survey with hematopoietic system cancer (such as：Grain/monocytic leukemia) in terms of detection in, provide important references for clinical diagnosis.
But it is associated with the monitoring of entity tumor, from the point of view of current patent and literature search, still lacks enough systematicness
Correlative study, and not yet form matured product, also there is no Patents mandate in CONTINENTAL AREA OF CHINA.
In view of this, it is special to propose the present invention.
The content of the invention
It is an object of the invention to provide a kind of flow cytometer detection kit for detecting tumor markers, to solve above-mentioned ask
Topic.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
A kind of flow cytometer detection kit for detecting tumor markers, including to mark the CD14 of Cell membrane antigens to resist
Body and CD16 antibody, and to mark the antibody of endochylema internal antigens；
The endochylema internal antigens are TKTL1 and/or DNASE1L1.
The present invention principle be, the CD14 gone out with CD14 and CD16 antibody labeling in object peripheral blood to be detected+CD16+
(resisted after cell, most of impurity of the removal in addition to leucocyte with antitumor mark X to be detected inside i.e. above-mentioned endochylema
It is former) the antibody labeling tumor markers；
Use flow cytomery X+CD14+CD16+Cell and CD14+CD16+Cell quantity and count the former with the latter
The ratio of cell quantity.
Flow cytometry (Flow Cytometry, FCM) be one kind on functional level to unicellular or other biological grain
The sub detection means for carrying out quantitative analysis and sorting, it can be with up to ten thousand cells of high speed analysis, and energy is while from a cell
Measure multiple parameters.By using flow cytometry to CD14+CD16+And X+CD14+CD16+The quantity of positive cell is examined
Survey, can quickly, precision evaluate global tumor markers expression.
Have determined the monocyte that there is different subtype in human peripheral blood at present.It is known it is main according to CD14 and
The expression of CD16 antigens is divided into 4 hypotypes：CD14++CD16-(G1 hypotypes), CD14++CD16+(G2 hypotypes), CD14+CD16-(G3
Hypotype), CD14+CD16+(G4 hypotypes).CD14+CD16+It is one of wherein topmost hypotype, it is thinner than the monokaryon of other hypotypes
Born of the same parents have stronger phagocytic activity, thus for the principle angle of the present invention, detect CD14+CD16+The monocyte of hypotype is more
It is meaningful.
Tumor markers (TM), refers to that characteristic is present in malignant cell, or produced extremely by malignant cell
Material, or the material that host produces to the stimulate the reaction of tumour, and can reflect tumorigenesis monitors tumour pair
One class material of therapeutic response.Work as CD14+CD16+When cell swallows tumour cell, tumor markers will be in CD14+CD16+Carefully
Born of the same parents are enriched with, and CD14+CD16+Cell be in people's body-internal-circulation campaign, thus can be by detecting CD14+CD16+Tumour in cell
Mark content carrys out the convenient Tumor Marker Levels characterized in human body.
The difference of tumour cell and normal somatic cell, it is main at two aspects：
Metabolism：Tumour cell is often shown to energy because its propagation is fast and is not regulated and controled by normal cell-cycle
The demand of the formula of hungering and thirst of quantity of material (glucose etc.), but due to the variation of its metabolic mechanism, metabolism particularly energetic supersession
Efficiency is often far below normal somatic cell, is utilized for example, fully effective metabolism can not be carried out to glucose, but a kind of waste
The consumption of formula.And enzyme material of the tumour cell required in this metabolic process, its expression quantity and normal somatic cell there is also
Huge difference, the break-through point that can be detected as tumour cell.
Apoptosis：Another feature that tumour cell is different from normal somatic cell is exactly to immortalize, and tumour cell is not
Regulated and controled by normal cell-cycle.Apoptosis Mechanism, which has had, more clearly to be studied, tumour cell not apoptosis, that certainty
Cause the expression quantity of regulating cell apoptosis or the factor played a key effect in apoptosis process, semiochemicals or enzyme
There is substantially exception, this can also serve as the break-through point of tumour cell detection.
Two tumor markerses preferred for this invention are related to metabolism with Apoptosis respectively.
TKTL1 (transketolase 1) is transketolase 1, it, the non-oxide way in main regulation glycometabolism approach
The developing stage of footpath, the high expression in kinds of tumors, its expression quantity and tumour, invasion and attack and transfer, and prognosis have substantial connection.
There is important suggesting effect in terms of nutrition arrangement to tumor patient and follow-up treatment.
DNASE1L1 (Deoxyribonuclease I-Linke 1) plays an important role in apoptosis process, but
In tumour cell, normal Apoptosis mechanism failure, DNASE1L1 activity is suppressed, but its great expression and in tumour cell
Accumulation.Its expression quantity can be used as the index for differentiating tumour cell or Apoptosis mechanism abnormal cell.
It is preferred that, flow cytometer detection kit as described above, the flow cytometer detection kit also include the CD14 antibody,
The corresponding fluorescence secondary antibody of one or more antibody in the antibody of CD16 antibody and endochylema internal antigens.
It is further preferred that the fluorescence secondary antibody is the antibody of F (ab') 2.
The present invention needs to mark CD14+CD16+TKTL1 and/or DNASE1L1 antigens inside cell cytosol, due to F
(ab') 2 antibody do not have Fc sections, and the ability of penetration cell film is stronger, and specificity is more preferable, thus the preferably antibody of F (ab') 2 is as glimmering
Light secondary antibody.
Meanwhile, in order to reduce operating procedure, it is preferred that the CD14 antibody, CD16 antibody and endochylema internal antigens it is anti-
Body is fluorescent labeled antibody, and fluorescence color is different.
Further, no matter for fluorescence secondary antibody or fluorescence primary antibody, it is preferred that the fluorescence labeling be PE, CY5,
Any of CY7, per-CP and TRITC.
It is preferred that, flow cytometer detection kit as described above, the flow cytometer detection kit also include with the endochylema
IgG antibody of the antibody of portion's antigen with Species origin.
Effect with the IgG antibody of Species origin is, can mark and the antibody non-specific binding of endochylema internal antigens
Non-specific material, thus can be deducted its signal as background signal, it is specifically shown in the embodiment of the present invention.
It is preferred that, flow cytometer detection kit as described above, the flow cytometer detection kit also include lysate, closing and
Punch any one or more in buffer solution, fixer, rinsing liquid.
It is further preferred that the lysate is working solution or its mother liquor containing each composition of following concentration：
8mM~12mM Tris-HCL, 8mM~12mM NaH2PO4And/or NaHPO4, 120mM~140mM NaCl, volume
Triton X-100,8mM that percentage is 0.7%~1.3%~12mM Na4P2O7·10H2O, pH=7.2~7.7.
It is further preferred that the closing and punching buffer solution are the working solution containing each composition of following concentration or its mother
Liquid：
0.008g/mL~0.012g/mL BSA and percentage by volume is molten for 0.7%~1.3% Triton X-100
Liquid, solvent is 1 × TBS or 1 × PBS solution.
It is further preferred that the fixer is working solution or its mother liquor containing each composition of following concentration：
With volume percent, the mixed solution containing 0.8%~1.2% formaldehyde and 0.33%~0.38% methanol,
Solvent for use is water.
It is further preferred that the rinsing liquid is the working solution or its mother liquor of each composition containing following concentration：
1 × PBS solution.
Compared with prior art, beneficial effects of the present invention are：
1), the invention provides detection tumour antigen kit, the kit be directed to blood in TKTL1 and
DNASE1L1 is detected, and then knows the expression of the global tumour antigen of object to be measured, thus available for the early stage of tumour
Diagnosis.
2), flow cytometry (Flow Cytometry, FCM) be one kind on functional level to unicellular or other biological
Particle carries out the detection means of quantitative analysis and sorting, and it can be with up to ten thousand cells of high speed analysis, and energy is simultaneously from a cell
In measure multiple parameters.Flow cytometry combine the present invention kit can quickly, evaluate to precision global tumor markers
Expression.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The accompanying drawing used required in embodiment or description of the prior art is briefly described, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is the partial results figure of flow cytometry；Figure 1A is total mononuclear cell, and the scope that Figure 1B R2 are outlined is
CD14+CD16+Cell；
Fig. 2 is the partial detection figure of embodiment 1；Fig. 2A is control group, and Fig. 2 B are DNASE1L1, and Fig. 2 C are TKTL1；
Fig. 3 is the partial detection figure of embodiment 2；Fig. 3 A are control group, and Fig. 3 B are DNASE1L1, and Fig. 3 C are TKTL1.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, be
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
1st, peripheral blood blood sample 1 is taken, 200ul is drawn, antibody anti-CD14-FITC (BD companies, article No. is added
555397) 40ul, anti-CD16-APC (BD companies, article No. 561304) 10ul, room temperature lucifuge is incubated 15min；
2nd, add 200ul fixative room temperatures lucifuge and be incubated 8min；The composition of the fixative is：Percentage by volume is
0.8% formaldehyde, 0.30% methanol that percentage by volume is；
3rd, add lysate 3mL into sample, the concussion that is vortexed is mixed, and lucifuge is incubated 15min, splitting erythrocyte；The cracking
Liquid contains 8mM Tris-HCL, 8mM NaH2PO4And/or NaHPO4, 140mM NaCl, percentage by volume be 1.3% Triton
X-100、8mM Na4P2O7·10H2O, pH=7.2；
4th, 700g centrifuges 6min, removes supernatant；Supernatant slowly is outwelled, residual liquid is drawn with paper, the concussion that is vortexed is resuspended thin
Born of the same parents；
5th, 80ul closings and punching buffer solution are added, thrum is mixed；
The Triton X-100 that the BSA and percentage by volume that closing and the punching buffer solution is 0.012g/mL are 1.3%
Solution；Solvent is 1 × TBS solution.
6th, above-mentioned solution system is bisected into two parts, be separately added into anti-TKTL1 (rabbit polyclonal,
Sigma, article No. HPA000505) and anti-DNASE1L1 antibody (rabbit polyclonal, sigma, article No.
HPA072635), thrum is mixed, and lucifuge is incubated 20min；The adding proportion of antibody is 1:1000；
7th, 2ml PBS (8g/L NaCl, 0.2g/L KCl, 1.4g/L Na are separately added into2HPO4、0.27g/L KH2PO4)
Wash, be vortexed and mix, 800g centrifugation 5min remove supernatant, wash 2~3 times, cell is resuspended after washing；
8th, 2ul anti-rabbit-PE antibody (donkey polyclonal, abcam companies, goods are separately added into
Number ab7007), lucifuge is incubated 10min；
9th, 2ml PBS are separately added into wash, is vortexed and mixes, 800g centrifugation 5min remove supernatant, washed 2~3 times, are resuspended after washing
Cell；
10th, cell, flow cytomery is resuspended with 500ul PBS respectively.
Embodiment 2
1st, peripheral blood blood sample 2 is taken, 200 are drawn, antibody anti-CD14-FITC (BD companies, article No. 555397) is added
40ul, anti-CD16-APC (BD companies, article No. 561304) 10ul, room temperature lucifuge is incubated 15min；
2nd, add 200ul fixative room temperatures lucifuge and be incubated 6min；The composition of the fixative is：Percentage by volume is
1.2% formaldehyde, 0.38% methanol that percentage by volume is；
3rd, add lysate 3mL into sample, the concussion that is vortexed is mixed, and lucifuge is incubated 15min, splitting erythrocyte；The cracking
Liquid contains 12mM Tris-HCL, 12mM NaH2PO4And/or NaHPO4, 120mM NaCl, percentage by volume be 0.7%
Triton X-100、12mM Na4P2O7·10H2O, pH=7.7；
4th, 900g centrifuges 4min, removes supernatant；Supernatant slowly is outwelled, residual liquid is drawn with paper, the concussion that is vortexed is resuspended thin
Born of the same parents；
5th, 120ul closings and punching buffer solution are added, thrum is mixed；
The Triton X-100 that the BSA and percentage by volume that closing and the punching buffer solution is 0.008g/mL are 0.7%
Solution；Solvent is 1 × PBS solution.
6th, above-mentioned solution system is bisected into two parts, be separately added into anti-TKTL1 (rabbit polyclonal,
Sigma, article No. HPA000505) and anti-DNASE1L1 antibody (rabbit polyclonal, sigma, article No.
HPA072635), thrum is mixed, and lucifuge is incubated 15min；The adding proportion of antibody is 1:1000；
7th, 2ml PBS (8g/L NaCl, 0.2g/L KCl, 1.4g/L Na are separately added into2HPO4、0.27g/L KH2PO4)
Wash, be vortexed and mix, 800g centrifugation 5min remove supernatant, wash 2~3 times, cell is resuspended after washing；
8th, 2ul anti-rabbit-PE antibody (donkey polyclonal, abcam companies, goods are separately added into
Number ab7007), lucifuge is incubated 10min；
9th, 2ml PBS are separately added into wash, is vortexed and mixes, 800g centrifugation 5min remove supernatant, washed 2~3 times, are resuspended after washing
Cell；
10th, cell, flow cytomery is resuspended with 500ul PBS respectively.
Result to embodiment 1~2 is detected.During statistics, first CD14 is outlined from all monocytes (Figure 1A)+
CD16+Cell (scope that Figure 1B, R2 are outlined)；Again CD14 is outlined from R2 frames+CD16+DNASE1L1+Or CD14+CD16+
TKTL1+Cell (Fig. 2 and Fig. 3)；Wherein, the way of control group (Fig. 2A and Fig. 3 A) is, by anti-TKTL1 or anti-
DNASE1L1 antibody replaces with the IgG antibody of same Species origin；Remaining step is consistent with experimental group.
The embodiment 1 of table 1 and the testing result of embodiment 2 (GCI values)
Wherein, detected value is respectively in form：
Control group：(CD14+CD16+IgG+÷CD14+CD16+)×1000；
DNASE1L1 groups：(CD14+CD16+DNASE1L1+÷CD14+CD16+)×1000；
TKTL1 groups：(CD14+CD16+TKTL1+÷CD14+CD16+)×1000；
End value is GCI (globle canceration index, global canceration index) value, and its algorithm is the inspection of each group
Measured value subtracts the detected value of control group.
The line of demarcation value of normal and disease determines that the data of early stage are by substantial amounts of clinical data
TKTL1：It is normal less than 119, there is tumor risk more than 119 points；
DNASE1L1：0-100 is normal, 100-130 suggestion complete detections, and more than 130 have tumor risk；
The synthesis GCI values that final result integrates two groups of TKTL1 and DNASE1L1 are considered.
Embodiment 3
1st, by peripheral blood blood sample 3,200ul is drawn, antibody anti-CD14-FITC (BD companies, article No. is added
555397) 40ul, anti-CD16-APC (BD companies, article No. 561304) 10ul, room temperature lucifuge is incubated 15min；
2nd, add 200ul fixative room temperatures lucifuge and be incubated 5min；The composition of the fixative is：Percentage by volume is 1%
Formaldehyde, 0.35% methanol that percentage by volume is；
3rd, add lysate 3mL into sample, the concussion that is vortexed is mixed, and lucifuge is incubated 15min, splitting erythrocyte；The cracking
Liquid contains 10mM Tris-HCL, 10mM NaH2PO4And/or NaHPO4, 130mM NaCl, percentage by volume be 1% Triton
X-100、10mM Na4P2O7·10H2O, pH=7.5；
4th, 800g centrifuges 5min, removes supernatant；Supernatant slowly is outwelled, residual liquid is drawn with paper, the concussion that is vortexed is resuspended thin
Born of the same parents；
5th, 100ul closings and punching buffer solution are added, thrum is mixed；
The Triton X-100 that the BSA and percentage by volume that closing and the punching buffer solution is 0.01g/mL are 1% are molten
Liquid；
6th, in above-mentioned solution system add fluorescence labeling anti-TKTL1 (antibody be purchased from rabbitpolyclonal,
Sigma, article No. HPA000505, oneself adds fluorescence labeling) and the anti-DNASE1L1 antibody of fluorescence labeling (antibody is purchased from
Rabbit polyclonal, sigma, article No. HPA072635, oneself adds fluorescence labeling), thrum is mixed, and lucifuge incubation 15~
20min；The adding proportion of antibody is 1:1000；Note the anti-TKTL1 of fluorescence labeling and the anti-of fluorescence labeling
DNASE1L1 antibody should color and anti-CD14-FITC and anti-CD16-APC (BD companies, article No. 561304) it is glimmering
Light color is different.
7th, 2ml PBS (8g/L NaCl, 0.2g/L KCl, 1.4g/L Na are separately added into2HPO4、0.27g/LKH2PO4)
Wash, be vortexed and mix, 800g centrifugation 5min remove supernatant, wash 2~3 times, cell is resuspended after washing；
8th, cell, flow cytomery is resuspended with 500ul PBS.
Experimental example
Peripheral blood blood sample 3 is taken to be bisected into four groups, every group 3 parts, with the method described in embodiment 1~3 and comparative example
Detected, every group in triplicate；The set-up mode of wherein comparative example is：Removed on the basis of embodiment 3 plus lysate splits
Solution red blood cell simultaneously centrifuges the step of going the removal of impurity, remaining operation be the same as Example 3 (ready-to-use whole blood is detected).
The testing result of table 2 (GCI values)
As can be known from the above table, the method detection stability described in embodiment 1~3 is preferable and reproducible, and comparative example three
The floating of secondary detection is larger, and end value is far below embodiment, thus it is speculated that be due to not go the removal of impurity the reason for possible, antibody is non-
Specific binding is more, causes CD14+CD16+Value rises, and the antibody of tumor markers may be specific better, so
CD14+CD16+X+Change is little, and then causes GCI value entire lowerings, causes false negative.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, but it will be understood by those within the art that：Its
The technical scheme described in foregoing embodiments can still be modified, or to which part or all technical characteristic
Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill
The scope of art scheme.
Claims (10)
1. a kind of flow cytometer detection kit for detecting tumor markers, it is characterised in that including to mark cell membrane surface to resist
Former CD14 antibody and CD16 antibody, and to mark the antibody of endochylema internal antigens；
The endochylema internal antigens are TKTL1 and/or DNASE1L1.
2. flow cytometer detection kit according to claim 1, it is characterised in that the flow cytometer detection kit also includes institute
State the corresponding fluorescence secondary antibody of one or more antibody in the antibody of CD14 antibody, CD16 antibody and endochylema internal antigens.
3. flow cytometer detection kit according to claim 2, it is characterised in that the fluorescence secondary antibody is the antibody of F (ab') 2.
4. flow cytometer detection kit according to claim 1, it is characterised in that the CD14 antibody, CD16 antibody and born of the same parents
The antibody for starching internal antigens is fluorescent labeled antibody, and fluorescence color is different.
5. flow cytometer detection kit according to claim 1, it is characterised in that the flow cytometer detection kit also include with
IgG antibody of the antibody of the endochylema internal antigens with Species origin.
6. the flow cytometer detection kit according to any one of Claims 1 to 5, it is characterised in that the flow cytometer detection reagent
Box also includes lysate, closing and punches any one or more in buffer solution, fixer, rinsing liquid.
7. flow cytometer detection kit according to claim 6, it is characterised in that the lysate is each containing following concentration
The working solution of composition or its mother liquor：
8mM~12mM Tris-HCL, 8mM~12mM NaH2PO4And/or NaHPO4, 120mM~140mM NaCl, volume basis
Triton X-100,8mM that number is 0.7%~1.3%~12mM Na4P2O7·10H2O, pH=7.2~7.7.
8. flow cytometer detection kit according to claim 6, it is characterised in that the closing and punching buffer solution be containing
The working solution or its mother liquor of following each composition of concentration：
0.008g/mL~0.012g/mL BSA and percentage by volume is 0.7%~1.3% TritonX-100 solution, solvent
For 1 × TBS or 1 × PBS solution.
9. flow cytometer detection kit according to claim 6, it is characterised in that the fixer is each containing following concentration
The working solution of composition or its mother liquor：
With volume percent, the mixed solution containing 0.8%~1.2% formaldehyde and 0.33%~0.38% methanol is used
Solvent is water.
10. flow cytometer detection kit according to claim 6, it is characterised in that the rinsing liquid is to contain following concentration
Each composition working solution or its mother liquor：
1 × PBS solution.
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| CN115372611A (en) * | 2022-07-18 | 2022-11-22 | 中山大学孙逸仙纪念医院 | Application of CD16+ fibroblasts in diagnosis, prevention and treatment of monoclonal antibody drug-resistant breast cancer |
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