CN103725648A - Novel CTC (circulating tumor cell) enrichment technology and preparation method of kit - Google Patents
Novel CTC (circulating tumor cell) enrichment technology and preparation method of kit Download PDFInfo
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Abstract
The invention discloses a novel CTC (circulating tumor cell) enrichment technology and a preparation method of a kit. The technology comprises the steps as follows: through permutation and combination of all possible CD45 isomer protein sequence information, designing antigens, thereby producing multiple antibodies, screening out one antibody capable of recognizing all CD45 isomers, coupling the antibody onto a magnetic bead, and further capturing all leukocytes from blood; collecting 10 ml of peripheral venous blood; removing red blood cells through lysis by an osmosis lysis buffer; mixing a designed specific antibody, the magnetic bead and the rest cell sap containing no red blood cells to separate leukocytes; and obtaining a cell population containing CTCs with higher concentration. According to the technology, a negative collection technology is adopted, so that the proportion of the CTCs is increased substantially, and the rapid and accurate detection is facilitated; meanwhile, a negative screening means is adopted, so that the CTCs are reserved without loss, and the fact that tumor diseases cannot be detected and the treatment for patients is delayed due to the loss of certain CTCs is prevented.
Description
Technical field
The present invention relates to medical field, be specially a kind of new circulating tumor cell beneficiation technologies and test kit preparation method.
Background technology
Circulating tumor cell (circulating tumor cells, CTCs) referring to spontaneously or because operation of diagnosis and treatment is discharged into the tumour cell of Peripheral Circulation by solid tumor or metastasis, is the major reason that postoperative recurrence and distant metastasis appear in malignant tumor patient.In recent years, along with updating of detection technique, CTCs detects as a kind of novel non-invasive diagnostic instrument, finds that in early days the using value of the aspects such as patient's postoperative recurrence and distant metastasis, assessment curative effect and prognosis has become the focus of clinical study.
Molecular biology and clinical studies show, the invasion and attack of tumour and micrometastasis probably just occur in early days tumorigenic, and the discovery of clinical tumor at present and diagnosis still highly depend on the inspection of iconography and blood serum tumor markers, be difficult to transfer or the recurrence of early discovery tumour, be also difficult to reflect in time curative effect.Therefore, early discovery micrometastasis is not only significant to the judgement of tumor recurrence and prognosis, and guiding clinical treatment is also had to very large value.Studies show that in a large number, the detection of circulating tumor cell (circulating tumor cells, CTCs) contributes to the micrometastasis of early discovery tumour, monitoring postoperative recurrence, assessment curative effect and prognosis, and selects suitable individualized treatment.
Circulating tumor cell is the tumour cell that enters human peripheral blood.To its separation and enrichment, will play important medicine effect for monitoring patient tumors situation.Current circulating tumor cell enrichment mainly adopts the method for forward sorting, by the tumour cell of mononuclearcell in antibody and magnetic bead or the direct sorting peripheral blood of flow cytometer, the method exists shortcoming: 1, the tumour cell quantity in peripheral blood mononuclear cell is extremely low, approximately 1,000,000/, weak output signal, analysis difficulty is large, easily causes error; 2, monoclonal antibody screening, selective power is limited, and the tumour cell of antibody None-identified is left in the basket, and affects detected result.
Summary of the invention
Technical problem solved by the invention is to provide a kind of new circulating tumor cell beneficiation technologies and test kit preparation method, to solve the problem proposing in above-mentioned background technology.
Technical problem solved by the invention realizes by the following technical solutions:
A new circulating tumor cell beneficiation technologies, comprises the following steps:
(1) by all possible CD45 isomer protein of permutation and combination sequence, obtain an antibody and can identify all CD45 isomer;
(2) by making Multiple Antibodies, thereby filter out the antibody that can identify all CD45 isomer, be coupled on magnetic bead, and then from blood, catch all white corpuscles, wherein antigen sequence includes the total length in territory, extracellular region, territory, the extracellular region total length of different exon splicings, be intended to filter out a kind of antibody and can identify their common section, why do not adopt separately orange albumen region to be because we want by the protein conformation of the outer part of more real analog cell film, directly with multiple CD45 isomer antigen, remove to screen our antibody, thereby the antibody obtaining can be at many kinds of isomer of Direct Recognition CD45 under true horizon,
(3) cell screening of the CD45+ being in the great majority in the tunica albuginea confluent monolayer cells being separated in blood is fallen, in the residual cells obtaining, circulating tumor cell proportion increases substantially, thereby can obtain fast the circulating tumor cell of high density, and fast and easy detects accurately.
A new circulating tumor cell test kit preparation method, comprises the following steps:
(1) gather peripheral blood 10ml in anticoagulant heparin pipe, add in infiltration lysate, at room temperature hatch;
Wherein permeating lysate becomes:
NH4C1
NaHCO3
EDTA
(2) centrifugal to carrying out;
(3) remove supernatant, by obtaining the aseptic physiological saline of cell precipitation, wash one time, again centrifugal, collect cell precipitation;
(4) according to Sulfo-NHS-LC-Biotin Biotinylation method, CD45 antibody is carried out to biotin labeling;
(5) add a small amount of aseptic PBS in cell precipitation, blow and beat gently with re-suspended cell, add biotin labeled CD45 antibody simultaneously, mix, at room temperature hatch;
(6) in mixture, add the magnetic bead of Streptavidin coupling, with pipettor piping and druming, mixed solution is mixed;
(7) use with the electromagnetic equipment of multistage probe and collect magnetic bead, can be combined with vitamin H with the Streptavidin of magnetic bead coupling high degree of specificity, on the magnetic bead of collecting by electromagnetic equipment, have a large amount of white corpuscles containing CD45 antigen;
(8) remaining PBS suspension is carried out centrifugal, collect circulating tumor cell precipitation;
(9) according to different experiment purposes, circulating tumor cell is done to further processing, and then generate a reagent box.
In sum, beneficial effect of the present invention:
The present invention adopts negative collection technique, the cell screening of the CD45+ being in the great majority in the tunica albuginea confluent monolayer cells being separated in blood is fallen, in the residual cells obtaining, circulating tumor cell proportion increases substantially, thereby can obtain fast the cell of high density circulating tumor cell, fast and easy detects accurately, simultaneously owing to having adopted negative screening means, circulating tumor cell is break-even to be remained, the loss that has prevented some circulating tumor cell causes tumor disease to be detected, and delays patient treatment.
Embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only the present invention's part embodiment, rather than whole embodiment.Based on the embodiment in the present invention, those of ordinary skills, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
A new circulating tumor cell beneficiation technologies, comprises the following steps:
(1) all possible CD45 isomer protein of permutation and combination sequence, by carefully analyzing, prepares corresponding plurality of antigens, thereby obtains Multiple Antibodies;
(2) by making Multiple Antibodies, thereby filter out the antibody that can identify all CD45 isomer, be coupled on magnetic bead, and then from blood, catch all white corpuscles, wherein antigen sequence includes the total length in territory, extracellular region, territory, the extracellular region total length of different exon splicings, be intended to filter out a kind of antibody and can identify their common section, why do not adopt separately orange albumen region to be because we want by the protein conformation of the outer part of more real analog cell film, directly with multiple CD45 isomer antigen, remove to screen our antibody, thereby the antibody obtaining can be at many kinds of isomer of Direct Recognition CD45 under true horizon,
(3) cell screening of the CD45+ being in the great majority in the tunica albuginea confluent monolayer cells being separated in blood is fallen, in the residual cells obtaining, circulating tumor cell proportion increases substantially, thereby can obtain fast the circulating tumor cell of high density, and fast and easy detects accurately.
A new circulating tumor cell test kit preparation method, comprises the following steps:
(1) gather peripheral blood 10ml in anticoagulant heparin pipe, add in 30ml infiltration lysate, 22 ℃ of room temperatures, hatch 10min;
Wherein permeating lysate composition is:
NH4C1150mM
NaHCO312mM
EDTA0.1mM
(2) under the rotating speed of 250g/1500rpm, infiltration lysate is carried out to centrifugal 5min;
(3) remove supernatant, by obtaining the aseptic physiological saline of cell precipitation 0.9%NaCl, wash one time, again centrifugal, collect cell precipitation;
(4) according to Sulfo-NHS-LC-Biotin Biotinylation method, CD45 antibody is carried out to biotin labeling;
(5) add a small amount of aseptic PBS in cell precipitation, blow and beat gently with re-suspended cell, add biotin labeled CD45 antibody simultaneously, making its final concentration is 0.1mg/ml, mixes, and at room temperature hatches 15min;
(6) in mixture, add the magnetic bead of Streptavidin coupling, with pipettor piping and druming, piping and druming more than 5 times, mixes mixed solution;
(7) use with the electromagnetic equipment of multistage probe and collect magnetic bead, can be combined with vitamin H with the Streptavidin of magnetic bead coupling high degree of specificity, on the magnetic bead of collecting by electromagnetic equipment, have a large amount of white corpuscles containing CD45 antigen;
(8), by centrifugal under the rotating speed of 250g remaining PBS suspension, collect circulating tumor cell precipitation;
(9) according to different experiment purposes, circulating tumor cell is done to further processing, and then generate a reagent box.
In addition, be to be understood that, although this specification sheets is described according to embodiment, but be not that each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should make specification sheets as a whole, and the technical scheme in each embodiment also can, through appropriately combined, form other embodiments that it will be appreciated by those skilled in the art that.
Claims (2)
1. a new circulating tumor cell beneficiation technologies, is characterized in that, comprises the following steps:
(1) analyze all possible CD45 isomer protein sequence, design multiple antigen, prepare multiple antibody;
(2) by making Multiple Antibodies, thereby filter out the antibody that can identify all CD45 isomer, be coupled on magnetic bead, and then from blood, catch all white corpuscles, wherein antigen sequence includes the total length in territory, extracellular region, territory, the extracellular region total length of different exon splicings, be intended to filter out a kind of antibody and can identify their common section, why do not adopt separately orange albumen region to be because we want by the protein conformation of the outer part of more real analog cell film, directly with multiple CD45 isomer antigen, remove to screen our antibody, thereby the antibody obtaining can be at many kinds of isomer of Direct Recognition CD45 under true horizon,
(3) cell screening of the CD45+ being in the great majority in the tunica albuginea confluent monolayer cells being separated in blood is fallen, in the residual cells obtaining, circulating tumor cell proportion increases substantially, thereby can obtain fast the circulating tumor cell of high density.
2. a new circulating tumor cell test kit preparation method, is characterized in that, comprises the following steps:
(1) gather peripheral blood 10ml in anticoagulant heparin pipe, add in infiltration lysate, at room temperature hatch;
Wherein permeating lysate composition is:
NH4C1
NaHCO3
EDTA
(2) to infiltration lysate, carry out centrifugal;
(3) remove supernatant, by obtaining the aseptic physiological saline of cell precipitation, wash one time, again centrifugal, collect cell precipitation;
(4) according to Sulfo-NHS-LC-Biotin Biotinylation method, CD45 antibody is carried out to biotin labeling;
(5) add a small amount of aseptic PBS in cell precipitation, blow and beat gently with re-suspended cell, add biotin labeled CD45 antibody simultaneously, mix, at room temperature hatch;
(6) in mixture, add the magnetic bead of Streptavidin coupling, with pipettor piping and druming, mixed solution is mixed;
(7) use with the electromagnetic equipment of multistage probe and collect magnetic bead, can be combined with vitamin H with the Streptavidin of magnetic bead coupling high degree of specificity, on the magnetic bead of collecting by electromagnetic equipment, have a large amount of white corpuscles containing CD45 antigen;
(8) remaining PBS suspension is carried out centrifugal, collect circulating tumor cell precipitation;
(9) according to different experiment purposes, circulating tumor cell is done to further processing, and then generate a reagent box.
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| CN105277697A (en) * | 2015-10-27 | 2016-01-27 | 上海芯超生物科技有限公司 | Method for measuring number of circulating epithelial tumor cells in blood |
| WO2016017755A1 (en) * | 2014-07-30 | 2016-02-04 | 日立化成株式会社 | Method for capturing rare cells in blood |
| WO2016017756A1 (en) * | 2014-07-30 | 2016-02-04 | 日立化成株式会社 | Method for capturing rare cells in blood |
| CN105891165A (en) * | 2014-11-04 | 2016-08-24 | 郝淮杰 | Method and kit for separating rare cells from peripheral blood |
| CN106046163A (en) * | 2016-06-24 | 2016-10-26 | 安徽未名细胞治疗有限公司 | Complete-human-derived anti-CD45 all-molecule IgG antibody and application thereof |
| CN106916725A (en) * | 2017-03-20 | 2017-07-04 | 东华大学 | A kind of micro-fluidic chip for embedding functionalized nano-fiber film and its application |
| CN106969964A (en) * | 2017-02-23 | 2017-07-21 | 宁波美晶医疗技术有限公司 | The negative enrichment method and kit of rare cell in a kind of blood based on micro-fluidic and immune Magneto separate |
| CN108548920A (en) * | 2018-02-28 | 2018-09-18 | 江苏医诺万细胞诊疗有限公司 | A kind of detection method for the kit detecting circulating tumor cell using immunomagnetic beads negative sense absorption joint flow cytometry |
| CN108761080A (en) * | 2018-08-16 | 2018-11-06 | 中国科学院合肥物质科学研究院 | One specific cells in body captures box |
| CN109856388A (en) * | 2018-11-29 | 2019-06-07 | 北京优迅医学检验实验室有限公司 | The catching method and capture kit of circulating tumor cell |
| WO2019129178A1 (en) * | 2017-12-29 | 2019-07-04 | 上海白泽医学检验所有限公司 | Composition containing anti-cd45 monoclonal antibody, and method for using same |
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| WO2016017755A1 (en) * | 2014-07-30 | 2016-02-04 | 日立化成株式会社 | Method for capturing rare cells in blood |
| WO2016017756A1 (en) * | 2014-07-30 | 2016-02-04 | 日立化成株式会社 | Method for capturing rare cells in blood |
| JPWO2016017755A1 (en) * | 2014-07-30 | 2017-04-27 | 日立化成株式会社 | Blood circulating cancer cell capture method |
| JPWO2016017756A1 (en) * | 2014-07-30 | 2017-04-27 | 日立化成株式会社 | Blood rare cell capture method |
| CN105891165A (en) * | 2014-11-04 | 2016-08-24 | 郝淮杰 | Method and kit for separating rare cells from peripheral blood |
| CN105891165B (en) * | 2014-11-04 | 2019-09-13 | 郝淮杰 | The method and kit of rare cell in a kind of separation peripheral blood |
| CN105277697A (en) * | 2015-10-27 | 2016-01-27 | 上海芯超生物科技有限公司 | Method for measuring number of circulating epithelial tumor cells in blood |
| CN106046163A (en) * | 2016-06-24 | 2016-10-26 | 安徽未名细胞治疗有限公司 | Complete-human-derived anti-CD45 all-molecule IgG antibody and application thereof |
| CN106969964A (en) * | 2017-02-23 | 2017-07-21 | 宁波美晶医疗技术有限公司 | The negative enrichment method and kit of rare cell in a kind of blood based on micro-fluidic and immune Magneto separate |
| CN106969964B (en) * | 2017-02-23 | 2020-02-18 | 宁波美晶医疗技术有限公司 | Negative phase enrichment method and kit for rare cells in blood based on micro-fluidic and immunomagnetic separation |
| CN106916725A (en) * | 2017-03-20 | 2017-07-04 | 东华大学 | A kind of micro-fluidic chip for embedding functionalized nano-fiber film and its application |
| WO2019129178A1 (en) * | 2017-12-29 | 2019-07-04 | 上海白泽医学检验所有限公司 | Composition containing anti-cd45 monoclonal antibody, and method for using same |
| CN109991410A (en) * | 2017-12-29 | 2019-07-09 | 上海白泽医学检验所有限公司 | A kind of composition and its application method containing anti-CD45 monoclonal antibodies |
| CN108548920A (en) * | 2018-02-28 | 2018-09-18 | 江苏医诺万细胞诊疗有限公司 | A kind of detection method for the kit detecting circulating tumor cell using immunomagnetic beads negative sense absorption joint flow cytometry |
| CN108761080A (en) * | 2018-08-16 | 2018-11-06 | 中国科学院合肥物质科学研究院 | One specific cells in body captures box |
| CN109856388A (en) * | 2018-11-29 | 2019-06-07 | 北京优迅医学检验实验室有限公司 | The catching method and capture kit of circulating tumor cell |
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