CN107576789A - A kind of lyophilized NK cell surface antigens quality-control product and preparation method thereof - Google Patents
A kind of lyophilized NK cell surface antigens quality-control product and preparation method thereof Download PDFInfo
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- CN107576789A CN107576789A CN201711071957.5A CN201711071957A CN107576789A CN 107576789 A CN107576789 A CN 107576789A CN 201711071957 A CN201711071957 A CN 201711071957A CN 107576789 A CN107576789 A CN 107576789A
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Abstract
The purpose of the present patent application, which is to provide, provides a kind of lyophilized NK cell surface antigens quality-control product and preparation method thereof, described to be marked with reference to product by periphery lymphocyte by surface or intracellular specific antibody, is directly detected available for flow cytometer.The reference product are in preparation process, after specific antigen Positive Cell Counts, the concentration of specific cells is demarcated, therefore it can not only give the detection of flow cytometer comparative counting to provide reference, and can absolute counting detection in be used as with reference to product or count reference, in particular for volumetric method calculate flow cytometer different time, distinct device, different personnel reference and check and correction.
Description
Technical field
The present application relates to a kind of lyophilized NK cell surface antigens quality-control product and preparation method thereof, belong to immunology detection
Technical field.
Background technology
Leukocyte differentiation antigen is that leucocyte (in addition to blood platelet, vascular endothelial cell etc.) is ripe different in normal differentiation
In pedigree (lineage) and different phase and activation process, the cell surface marker that appears or disappears.They are mostly to wear film
Albumen or glycoprotein, outskirt containing after birth, transmembrane domains and cytoplasmic domain.Cluster differentiation antigen (cluster of
Differentiation, CD) for T cell during differentiation and maturation, the lymphocyte of different stages of development and different subclass can
Different differentiation antigens is expressed, this is to discriminate between the important symbol of lymphocyte.
FCM analysis method is a kind of analysis method of high-throughout cell or particulate, and it is mainly used in cell
The analysis of character, memebrane protein and intracellular organic matter.It is widely used in clinical diagnosis and scientific research field.Wherein in human peripheral
CD3-/CD16+/CD56+ cells are NKT (NK) cell, its clinical meaning determined：NK cells are natural killer cells,
Its target cell mainly has some tumour cells (including part cell line), virus infected cell, some autologous tissue's cell (such as blood
Cell), parasite etc., therefore NK cells are that body is antitumor, anti-infectious important immune factor, can also synthesize and secrete more
Kind of cell factor, plays that regulation is immune and hemoposieis, and as a kind of means of immune cell therapy, it is super quick anti-to participate in Type-II
Should and graft-versus-host reaction, sense organ transplanting after receptor response.
There is used quality-control product when flow cytometry is used for clinical detection at present：1. whole blood (such asCDChexWith)；2. liquid nitrogen cryopreservation cell etc..These objects of reference are
It is widely used, but problems with is present：Storage and transportation conditions harshness is, it is necessary to low temperature (2-8 DEG C) or ultralow temperature (- 80
℃)；Storage cycle is short, and the pot-life of whole blood is 3 months；Need first to carry out using process complexity, such as liquid nitrogen cryopreservation cell
Cell recovery.Same to cannot be used for different equipment or detection platform with reference to product, the data between a distinct device can not be entered
Row control.Therefore, offer one kind is provided badly and is easy to data acquisition, lower-cost check and correction product/reference product.
The content of the invention
In order to solve problems of the prior art, the purpose of the present patent application is to provide a kind of lyophilized NK cell surfaces
Antigen quality-control product and preparation method thereof.
Specifically, the preparation method of the lyophilized NK cell surface antigens quality-control product described in the present patent application, including it is as follows
Step：
1. collecting human peripheral blood cell, by density gradient centrifugation, human peripheral lymphocyte is collected；
2. rinsed using the phosphate buffer containing serum or Chinese krebs solution (Hank ' s buffer)；
3. adding anti-human CD3 and anti-human CD16, CD56 antibody, lucifuge, it is incubated, by centrifugation, cell is collected, again in phosphorus
Suspension cell in phthalate buffer or Hank ' s liquid, elutes uncombined Excess antibody, the cell of antibody labeling is harvested by centrifugation；
4. by the cell of harvest, fixer is added, using the mode of concussion or piping and druming, after fully mixing, lucifuge is fixed；
5. eluting fixer, using flow cytometer, positive cell number is counted, according to count results, in cell frozen-dried protective
In buffer solution, positive cell is diluted；
6. it will be freezed after the solution packing containing cell；
It is white or yellowish toner, pie to bottle content 7. vacuumizing lyophilized；
8. being taken out from freeze dryer, Quick seal, preserve.
Further, in the step 1), through venous blood collection or buffy coat (Buffy coat), collector are rinsed
Peripheral blood cells, cell suspension is laid in the human lymphocyte separating liquid of sucrose solution or commercialization, centrifuged so that no
With the cell aggregation of density, human peripheral lymphocyte is collected.
Further, in the step 3), anti-human CD3 and anti-human CD16, CD56 antibody, lucifuge, 15-30 DEG C of incubation are added
15-30 minutes, by centrifugation, cell is collected, again the suspension cell in phosphate buffer or Hank ' s liquid, elution is uncombined
Excess antibody, the cell of antibody labeling is harvested by centrifugation.
Further, in the step 4), by the cell of harvest, according to 103-108The concentration of individual cells/ml, add solid
Determine liquid, using the mode of concussion or piping and druming, after fully mixing, at 2-30 DEG C, lucifuge, fix 0.25-48 hours.
Further, in the step 5), dilution positive cell concentration is 102-108Individual cells/ml.
Further, in the step 6), the solution containing cell is distributed into 0.2-2 milliliters/bottle, is placed in -80 to -20
DEG C, freeze 4-72 hours.
Further, in the step 7), lyophilized 12-96 hours are vacuumized.
Further, in the step 8), take out, Quick seal, preserved under the conditions of -20-8 DEG C from freeze dryer.
The present patent application also provides the lyophilized NK cell surface antigens quality-control product being prepared according to the above method.
The beneficial effect of the present patent application is：
1. the positive lyophilized cells of preliminary making can preserve 4-6 under conditions of -20 DEG C, in the product of same batch
Part need not carry out control checking, and the control that the different batches of product for each two moon needing to carry out is saved compared to whole blood is tested
Card；
2. the transport in non high temperature area, the positive lyophilized cells of preliminary making do not need cold chain, relative to liquid nitrogen cryopreservation cell
It is required strict in -80 DEG C of conditions, transport and storage cost are saved, can also meet the needs of more multi-terminal user；
3. the conventional FITC of mark fluorescent element selection used in lyophilized cells, PE, APC etc., it is currently normal that can be applied to
Advise the instrument of the different model from different manufacturers used.
4. lyophilized cells can also be used between different machines, different operating person or different experiments room for detection method, behaviour
Make the control of code and parameter setting.Testing result has comparativity and certain versatility.
5. product of the present invention designs for " flow cytometry ", the positive cell surface antigen of fluorescence antibody mark is through fixation
Stable quantity and fluorescence intensity are kept afterwards, compared to the cell dried frozen aquatic products for not doing fluorescence antibody mark, there is more preferable antigen table
Up to stability and cell characteristics reappearance.
Embodiment
Nonrestrictive explanation is carried out to the technical scheme described in the present patent application below in conjunction with specific embodiment.
Embodiment 1
The present patent application provides the preparation method of lyophilized NK cell surface antigen quality-control products, as described below：
1. through venous blood collection, human peripheral blood cell is collected, cell suspension is laid on sucrose solution, centrifuged so that no
With the cell aggregation of density, human peripheral lymphocyte is collected；
2. rinsed using the phosphate buffer containing serum；
3. adding anti-human CD3 and anti-human CD16, CD56 antibody, lucifuge, 15 DEG C are incubated 30 minutes, by centrifugation, collect thin
Born of the same parents, the suspension cell in phosphate buffer, elutes unlabelled Excess antibody again, and the cell of antibody labeling is harvested by centrifugation；
4. by the cell of harvest, according to 103The concentration of individual cells/ml, fixer is added, it is fully mixed using concussion mode
After even, at 5 DEG C, lucifuge, 36 hours are fixed；
5. eluting fixer, using flow cytometer, positive cell number is counted, according to count results, in cell frozen-dried protective
In buffer solution, dilution positive cell concentration is 105Individual cells/ml；
6. the solution containing cell is distributed into 0.2 milliliter/bottle, -80 DEG C are placed in, is freezed 4 hours；
Vacuumized lyophilized 12 hours 7. vacuumizing and freezing, be white or yellowish toner, pie to bottle content；
8. being taken out from freeze dryer, Quick seal, preserved under the conditions of -20 DEG C.
Embodiment 2
The present patent application provides the preparation method of Lyophilized Human NK lymphocyte quality-control products, as described below：
1. through rinsing buffy coat (Buffy coat), human peripheral blood cell is collected, cell suspension is laid in commodity
In the human lymphocyte separating liquid of change, centrifugation so that the cell aggregation of different densities, collect human peripheral lymphocyte；
2. rinsed using Chinese krebs solution (Hank ' s buffer)；
3. adding anti-human CD3 and anti-human CD16, CD56 antibody, lucifuge, 30 DEG C are incubated 15 minutes, by centrifugation, collect thin
Born of the same parents, the suspension cell in Hank ' s liquid, elutes unlabelled Excess antibody, the cell of antibody labeling is harvested by centrifugation again；
4. by the cell of harvest, according to 108The concentration of individual cells/ml, adds fixer, and the mode of piping and druming fully mixes
Afterwards, at 30 DEG C, lucifuge, 1 hour is fixed；
5. eluting fixer, using flow cytometer, positive cell number is counted, according to count results, in cell frozen-dried protective
In buffer solution, dilution positive cell concentration is 108Individual cells/ml；
6. the solution containing cell is distributed into 2 milliliters/bottle, -20 DEG C are placed in, is freezed 72 hours；
It is white or yellowish toner, pie to bottle content 7. vacuumizing lyophilized 12 hours；
8. being taken out from freeze dryer, Quick seal, preserved under the conditions of -20 DEG C.
Experimental example 1
The people periphery CD3-/CD16+/CD56+ cells that two different batches freeze preliminary making respectively take 3 different samples,
20 tests are carried out, every microlitre of CD3 feminine gender is obtained and CD16, CD56 Positive Cell Counts is as shown in following table.Display is lyophilized
People's peripheral T lymphocyte of preliminary making can keep preferable uniformity between different operators and laboratory, as a result such as
Shown in following table：
Table -1
As a result show：
1. cell prepared by this method, keeps complete cellular morphology and cellular content, or phase similar to fresh cells
Together；
2nd, cell prepared by this method, maintains surface antigen expression quantity, does not have target antigen Loss；
3rd, positive cell prepared by this method, testing result is consistent on different flow cytometers, can form different machines
Reference and control between type；
4th, positive cell prepared by this method, quantity is homogeneous, calibration and reference available for streaming counting.
Lyophilized NK cell surface antigens quality-control product that the present patent application provides and preparation method thereof, is marked using fluorescence antibody
Positive lyophilized cells 4-6 can be preserved under conditions of -20 DEG C, every batch can be carried out and largely prepared, use is same
The certified products of batch need not carry out control checking, and compared to the whole blood that storage life only has 3 months, it is substantial amounts of to save user
Compare the work of checking.The product of the present invention can preserve more than 2 years at 2-8 DEG C, and 8-30 DEG C can preserve more than 6 months, therefore transport
Defeated process does not need cold chain transportation, the condition for being not higher than -80 DEG C required for solving in frozen cell transportation, saves fortune
Defeated cost and energy resource consumption, user's acquisition of being more convenient for.Product of the present invention designs for " flow cytometry ", fluorescence antibody mark
Positive cell surface antigen keeps stable quantity and fluorescence intensity after fixation, compared to the cell for not doing fluorescence antibody mark
Dried frozen aquatic products, there are more preferable antigen presentation stability and cell characteristics reappearance.Antibody fluorescence element selection used in lyophilized cells
Conventional FITC, PE, APC etc., it can be applied to the instrument from different manufacturers, different model at present.Lyophilized cells also are used as
The different experiments room of control between different machines, different operating person or to(for) detection method, operational procedure and parameter setting.Detection
As a result there is comparativity and certain versatility.During NK cells are as immune cell therapy, immunocyte, which is fed back, to advance
Row NK cell numbers are detected, and testing result need to have to be compared with reference to product.
The present invention is described in detail for the general explanation of the above and specific embodiment, but on the basis of the present invention,
It can be made some modifications or improvements, will be apparent to those skilled in the art.Therefore, without departing from the present invention
These modifications or improvements on the basis of spirit, belong to the scope of protection of present invention.
Claims (9)
1. a kind of preparation method of lyophilized NK cell surface antigens quality-control product, it is characterised in that comprise the following steps：
1) human peripheral blood cell is collected, by density gradient centrifugation, cell suspension is tiled, centrifuged, collector periphery lymph is thin
Born of the same parents；
2) rinsed using the phosphate buffer containing serum or Chinese krebs solution (Hank ' s buffer)；
3) anti-human CD3 and anti-human CD16, CD56 antibody are added, lucifuge, is incubated, by centrifugation, cell is collected, again in phosphate
Suspension cell in buffer solution or Hank ' s liquid, more and antibody is eluted, the cell after mark is harvested by centrifugation；
4) by the cell of harvest, fixer is added, after fully mixing, lucifuge is fixed；
5) fixer is eluted, using flow cytometer, counts positive cell number, according to count results, is buffered in cell frozen-dried protective
In liquid, positive cell is diluted；
6) will be freezed after the solution packing containing cell；
7) vacuumize lyophilized, be white or yellowish toner, pie to bottle content；
8) take out, Quick seal, preserve from freeze dryer.
2. the preparation method of lyophilized NK cell surface antigens quality-control product according to claim 1, it is characterised in that：The step
It is rapid 1) in, through venous blood collection or rinse buffy coat, collect human peripheral blood cell, cell suspension is laid in sucrose solution
Or in the human lymphocyte separating liquid of commercialization, centrifugation so that the cell aggregation of different densities, collector periphery lymph are thin
Born of the same parents.
3. the preparation method of lyophilized NK cell surface antigens quality-control product according to claim 1, it is characterised in that：The step
It is rapid 3) in, add anti-human CD3 and anti-human CD16, CD56 antibody, lucifuge, 15-30 DEG C of incubation 15-30 minute, by centrifugation, collect
Cell, the suspension cell in phosphate buffer or Hank ' s liquid, elutes uncombined Excess antibody, antibody is harvested by centrifugation again
The cell of mark.
4. the preparation method of lyophilized NK cell surface antigens quality-control product according to claim 1, it is characterised in that：The step
It is rapid 4) in, by the cell of harvest, according to 103-108The concentration of individual cells/ml, add fixer, using concussion or piping and druming
Mode, after fully mixing, at 2-30 DEG C, lucifuge, fix 0.25-48 hours.
5. the preparation method of lyophilized NK cell surface antigens quality-control product according to claim 1, it is characterised in that：The step
It is rapid 5) in, dilution positive cell concentration is 102-108Individual cells/ml.
6. the preparation method of lyophilized NK cell surface antigens quality-control product according to claim 1, it is characterised in that：The step
It is rapid 6) in, the solution containing cell is distributed into 0.2-2 milliliters/bottle, is placed in -80 to -20 DEG C, freezes 4-72 hours.
7. the preparation method of lyophilized NK cell surface antigens quality-control product according to claim 1, it is characterised in that：The step
It is rapid 7) in, vacuumize lyophilized 12-96 hours.
8. the preparation method of lyophilized NK cell surface antigens quality-control product according to claim 1, it is characterised in that：The step
It is rapid 8) in, take out, Quick seal, preserved under the conditions of -20-8 DEG C from freeze dryer.
9. the lyophilized NK obtained by the preparation method of the lyophilized NK cell surface antigens quality-control product according to claim 1-8 is thin
Cellular surface antigen quality-control product.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108663510A (en) * | 2018-04-28 | 2018-10-16 | 璞晞（广州）生物免疫技术有限公司 | The check and evaluation method and kit of hepatopathy NK cell functions |
| CN111528219A (en) * | 2020-05-13 | 2020-08-14 | 上海市计量测试技术研究院 | Freeze-drying protective agent for T lymphocyte subpopulation counting standard substance and application thereof |
| WO2021012925A1 (en) * | 2019-07-24 | 2021-01-28 | 泛肽生物科技(浙江)有限公司 | Method for measuring human peripheral blood lymphocytes |
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| CN101358968A (en) * | 2008-09-05 | 2009-02-04 | 甘肃省医学科学研究院 | Quality control quality of freeze-dried human lymphocyte surface antigen and method for preparing same |
| CN103923879A (en) * | 2014-04-29 | 2014-07-16 | 湖北华赛生物医药技术有限公司 | Preparation method and application of NK cytokine mixture |
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2017
- 2017-11-03 CN CN201711071957.5A patent/CN107576789A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101358968A (en) * | 2008-09-05 | 2009-02-04 | 甘肃省医学科学研究院 | Quality control quality of freeze-dried human lymphocyte surface antigen and method for preparing same |
| CN103923879A (en) * | 2014-04-29 | 2014-07-16 | 湖北华赛生物医药技术有限公司 | Preparation method and application of NK cytokine mixture |
Non-Patent Citations (1)
| Title |
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| 刘映霞 等: ""抗NKG2D多克隆抗体抑制NK和LAK细胞细胞毒效应的研究"", 《细胞与分子免疫学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108663510A (en) * | 2018-04-28 | 2018-10-16 | 璞晞（广州）生物免疫技术有限公司 | The check and evaluation method and kit of hepatopathy NK cell functions |
| WO2021012925A1 (en) * | 2019-07-24 | 2021-01-28 | 泛肽生物科技(浙江)有限公司 | Method for measuring human peripheral blood lymphocytes |
| CN111528219A (en) * | 2020-05-13 | 2020-08-14 | 上海市计量测试技术研究院 | Freeze-drying protective agent for T lymphocyte subpopulation counting standard substance and application thereof |
| CN111528219B (en) * | 2020-05-13 | 2022-03-15 | 上海市计量测试技术研究院 | Freeze-drying protective agent for T lymphocyte subpopulation counting standard substance and application thereof |
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