CN109187710A - A kind of CD133 mass spectrum streaming antibody, preparation method and application - Google Patents
A kind of CD133 mass spectrum streaming antibody, preparation method and application Download PDFInfo
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- CN109187710A CN109187710A CN201811055078.8A CN201811055078A CN109187710A CN 109187710 A CN109187710 A CN 109187710A CN 201811055078 A CN201811055078 A CN 201811055078A CN 109187710 A CN109187710 A CN 109187710A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5073—Stem cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Abstract
The invention belongs to field of biotechnology, specifically disclose a kind of CD133 mass spectrum streaming antibody, preparation method and application, the CD133 mass spectrum streaming antibody is using lanthanide series metal solution and the pure antibody of CD133 as raw material, by what is be prepared after on lanthanide series metal load to the pure antibody of CD133.The preparation method of the antibody is the following steps are included: prepare metal load object solution, and CD133 antibody restores, purified metal load, purifying CD133 antibody, preparation CD133 antibody complex, cleaning CD133 antibody complex and etc..CD133 mass spectrum streaming antibody body made of the present invention has filled up the blank of the prior art, and the antibody of mass spectrum flow cytometer detection can be used as the marker of mass spectrum streaming research stem cell, has expanded the new field of mass spectrum streaming technology research stem cell.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of CD133 mass spectrum streaming antibody, preparation method and application.
Background technique
Streaming technology is most classic Single cell analysis technology, can carry out multi-parameter detection to cell.Conventional flow makes
Cell surface marker is detected using fluorescence light path with fluorophor coupled antibody.Traditional streaming technology is based on fluorescence detection
System, but due to the overlapping of different fluorophor emission spectrum, the signal that will lead to interchannel is interfered with each other, and works as sense channel
When more than 8, compensation adjustment is highly dependent on artificial experience, so that experimental design and compensation calculation then become very multiple
It is miscellaneous.
The mass spectrum streaming technology of present emerging development distinguishes cell by mass-spectrometric technique using metallic element coupled antibody
Form.The detection system and tag system of novel mass spectrum streaming are different from conventional flow technology, and traditional streaming technology is
The mass spectrum streaming technology novel based on fluorescence detecting system uses metallic element as label in conjunction with antibody；Conventional flow skill
For art using laser and electron multiplier as detection means, novel mass spectrum streaming uses ICP mass-spectrometric technique as detection hand
Section.Novel mass spectrum streaming technology thoroughly solves fluorescence benefit by being combined to traditional streaming technology and mass-spectrometric technique
Problem is repaid, and realizes most 130 marks while detecting, and the BD flow cytometer of most top configuration now, at most simultaneously
Detect 50 marks.Its powerful data retrieval capabilities and bioinformatics of mass spectrum streaming are closely coupled, to original data into
Depth excavation is gone.
CD133 antibody is often expressed in immature candidate stem cell or progenitor cells, it has been reported that it is in brain tumor, forefront
Gland cancer, the Lewis lung cancer of mouse, B16 melanoma stem cell in have expression, have been considered as these tumor stem cells
One of mark.Discovery has been reported, discovery has the expression of two kinds of antigen mRNA of CD133 in kinds of tumor cells system.CD133-2
The mRNA of antigen is expressed in the cell surfaces height such as lung cancer cell line, pancreatic carcinoma and breast cancer cell line, and CD133-1
The mRNA high of antigen be expressed in retinoblastoma cell cancerous cell line and with its identical cancer cell surfaces that originate from.
As stem cell specific surface antigen, the mRNA of CD133 can be detected in a variety of cancerous cell lines, be prompted
CD133 is a kind of broad-spectrum marker of tumor stem cell, is a kind of unique stem-cell marker, and molecular weight 120kDa has 5
A transmembrane region, is once named as AC133.2000, in the 7th human leucocyte differentiation antigen state that Britain Harrogate is held
Border working conference (7th International Workshop on Human Leucocyte Differentiation
Antigens, HLDA7) on be officially named CD133.Can be used for screening some tumor stem cells not yet sub-elected or into
One step purifies the tumor stem cell sub-elected.
However since mass-spectrometric technique is the technology of a newborn development, the commercially available mass spectrum streaming monoclonal antibody of existing market is only
300 kinds or so, most of mass spectrum streaming antibody all needs voluntarily to synthesize, and lacks the antibody of the CD133 mass spectrum streaming of finished product, limits
Application of the CD133 as tumor stem cell broad-spectrum marker.
Summary of the invention
A kind of CD133 mass spectrum streaming antibody provided by the invention, preparation method and application, the CD133 mass spectrum streaming antibody
The blank of the prior art is filled up, the antibody of mass spectrum flow cytometer detection has a good application prospect.
The present invention provides a kind of CD133 mass spectrum streaming antibody, the CD133 mass spectrum streaming antibody is molten with lanthanide series metal
Liquid and the pure antibody of CD133 are raw material, by what is be prepared after on lanthanide series metal load to the pure antibody of CD133.
The present invention provides a kind of preparation methods of CD133 mass spectrum streaming antibody, comprising the following steps:
S1 prepares metal load object solution: preparing lanthanide series metal solution, 37 DEG C of incubation 30-40min obtain metal load
Object solution；
The reduction of S2, CD133 antibody: it is pure anti-that R-Buffer and CD133 is added into molecular cut off 50kDa centrifugal ultrafiltration pipe
Body mixes, and centrifugation abandons supernatant, TCEP-R-Buffer is added into obtained precipitating, mixes, and 37 DEG C of incubation 15-30min are obtained
To the antibody-solutions of reduction；
Wherein, the preparation method of the TCEP-R-Buffer of every 1000 μ L is as follows: being added into 8 μ L 0.5M TCEP solution
992 μ L R-Buffer are mixed；
Purified metal load: S3 is added L-Buffer into molecular cut off 3kDa centrifugal ultrafiltration pipe, adds S1 and obtain
The metal load object solution arrived mixes, and centrifugation continuously adds C-Buffer, mixes, centrifugation, recycling precipitating；
S4 purifies CD133 antibody: C-Buffer is added in the antibody-solutions obtained to S2, is centrifuged, abandons supernatant, then again
C-Buffer is added to mix, centrifugation, abandons supernatant, the precipitating of recycling, this is precipitated as the CD133 antibody of purifying；
S5 prepares CD133 antibody complex: the precipitating recycled in S3 is resuspended with C-Buffer, and will be in re-suspension liquid and S4
The CD133 antibody of obtained purifying mixes, and 37 DEG C of incubation 60-120min obtain CD133 antibody complex；
S6, clean CD133 antibody complex: with W-Buffer clean S5 obtained in CD133 antibody complex, done
Net CD133 mass spectrum streaming antibody；
Wherein the effect of R-Buffer is antibodies buffer；The effect of L-Buffer and C-Buffer is lanthanide series metal buffering
Liquid；The effect of W-Buffer is cleaning solution.
Preferably, in the preparation method of above-mentioned CD133 mass spectrum streaming antibody, in S1, the end of the lanthanide series metal solution is dense
Degree is 2.5mM, and the lanthanide series metal is ytterbium chloride or lanthanum chloride.
Preferably, in the preparation method of above-mentioned CD133 mass spectrum streaming antibody, in S2, the pure antibody of R-Buffer:CD133:
The volume ratio of TCEP-R-Buffer is 300:100-200:100.
Preferably, in the preparation method of above-mentioned CD133 mass spectrum streaming antibody, in S3, metal load object solution, L-Buffer
Volume ratio with C-Buffer is 100:200:300.
Preferably, in the preparation method of above-mentioned CD133 mass spectrum streaming antibody, in S4, the C-Buffer that is added for the first time, the
The volume ratio for the TCEP-R-Buffer being added in the C-Buffer and S2 of secondary addition is 300:400-500:100.
Preferably, in the preparation method of above-mentioned CD133 mass spectrum streaming antibody, specific step is as follows by S6: obtaining to S5
300 μ L W-Buffer are added in 50kDa centrifugal ultrafiltration pipe containing CD133 antibody complex, mix, 12,000g centrifugations
10min abandons supernatant, then washs precipitating three times with 400 μ L W-Buffer, is eventually adding 50 μ L W-buffer, rinses, and collects super
Solution A in chimney filter；Then the centrifugal ultrafiltration pipe 1000g is centrifuged 2min, then rinses tube wall with 50 μ L W-buffer, collected super
Solution B in chimney filter merges solution A and solution B in super filter tube, obtains clean CD133 mass spectrum streaming antibody.
The present invention provides above-mentioned CD133 mass spectrum streaming antibody answering as mass spectrum flow cytometer detection stem cell markers
With.
The present invention provides the preparation methods of above-mentioned CD133 mass spectrum streaming antibody to prepare mass spectrum flow cytometer detection stem cell
Application in marker.
Compared with prior art, a kind of CD133 mass spectrum streaming antibody provided by the invention, preparation method and application, have
Below the utility model has the advantages that
Before this invention, the CD133 mass spectrum streaming antibody of lanthanide series metal load is not present in mass spectrum streaming antibody, in mass spectrum
Stem cell is studied in streaming research, and such significant CD133 mass spectrum streaming antibody is lacked in cell getting up early differentiation, limits mass spectrum
The application range of streaming technology, the present invention synthesize the CD133 mass spectrum streaming antibody of new lanthanide series metal load by structure, can be in institute
Have in living cells and express, and expression quantity is high, has filled up the blank in market, has provided to mass spectrum flow cytometer detection stem-cell marker new
Target antibody has expanded the new field of mass spectrum streaming technology research stem cell.
Detailed description of the invention
Fig. 1 is that the CD133 mass spectrum streaming antibody (abbreviation 172Yb_CD133 (v) in figure) of the embodiment of the present invention does not set circle
The expression of door；
Wherein Figure 1A is the whole expression for not setting circle door 172Yb_CD133 (v), and Figure 1B is TSNE1 multidimensional data
The channel case generated after dimensionality reduction, Fig. 1 C are the channel cases generated after TSNE2 multidimensional data dimensionality reduction.
Fig. 2 is the CD133 mass spectrum streaming antibody (abbreviation 172Yb_CD133 (v) in figure) of the embodiment of the present invention in living cells
In expression；
Wherein Fig. 2A is the whole expression of 172Yb_CD133 (v), and Fig. 2 B is generated after TSNE1 multidimensional data dimensionality reduction
Channel case, Fig. 2 C is the channel case generated after TSNE2 multidimensional data dimensionality reduction.
Fig. 3 is the background that the CD133 mass spectrum streaming antibody test of the embodiment of the present invention is；
Wherein Fig. 3 A is the Background in single living cell detection, and Fig. 3 B is the background in the detection of all living cells
Figure.
Specific embodiment
The present invention is described in detail combined with specific embodiments below, but should not be construed as limitation of the invention.It is following
Experimental method in embodiment is unless otherwise specified conventional method, the materials, reagents and the like used in the following examples, such as
Without specified otherwise, it is commercially available.
It is used in following embodimentsAntibody Labeling Kit (4rxn) is purchased from the U.S.
Fu Luda, including R-Buffer 6mL, C-Buffer 5.5mL, L-Buffer 1.4mL, W-Buffer8mL andPolymer (4tubes), Lanthanide solution (20 μ L) (i.e. 20 μ L of lanthanide series metal solution, label
Be ytterbium Yb, lanthanide series metal is ytterbium chloride)；Wherein the effect of R-Buffer is antibodies buffer；L-Buffer and C-Buffer
Effect be lanthanide series metal buffer；The effect of W-Buffer is cleaning solution.
The IgG (being purchased from Shanghai Yuan Mu Biotechnology Co., Ltd) of purifying: non-loaded (not contain BSA, protein, gelatin
Equal stabilizers)；The pure antibody of CD133 (unmarked, carrier-free), the production of German Mei Tian Ni company.
Consumptive material used in following embodiments has: the centrifugal ultrafiltration pipe of molecular cut off 3kDa, molecular cut off 50kDa
Centrifugal ultrafiltration pipe and streaming loading pipe, are commercially available.
Embodiment 1
In the embodiment of the present invention, CD133 mass spectrum streaming antibody be using lanthanide series metal solution and the pure antibody of CD133 as raw material,
By what is be prepared after on lanthanide series metal load to the pure antibody of CD133.Preparation method is as follows:
S1 prepares metal load object solution: the lanthanide series metal solution of final concentration of 2.5mM prepared, is mixed, 37 DEG C of incubations
30-40min obtains metal load object solution；Concrete operations are as follows:
It pre-processes lanthanide series metal: the lanthanide series metal solution of purchase (is contained into 20 μ L lanthanums in the lanthanide series metal solution conduit of purchase
It is metal chlorination ytterbium) 12,000g centrifugation 10s, it is ensured that reagent abandons supernatant in centrifugal ultrafiltration bottom of the tube, and 95 μ L L-Buffer are added
(phosphate buffer, pH7.0,1mol/l) is resuspended, and mixes, obtains to reaction tube；
It prepares metal load object solution: to the lanthanide series metal solution bought to which 5 μ L are added in reaction tube, obtaining end
Concentration is the lanthanide series metal solution of 2.5mM, is mixed, and 37 DEG C of incubation 30-40min obtain metal load object solution, incubation period is opened
Beginning S2 step.
The reduction of S2, CD133 antibody: being that 300 μ L R- are added in 50kDa centrifugal ultrafiltration pipe (Bai Zhirui) to molecular cut off
The pure antibody of CD133 (the great Bioisystech Co., Ltd in Shanghai) of Buffer and 100 μ L mixes, room temperature 12,000g centrifugation
10min abandons supernatant, 100 μ L 4mM TCEP-R-Buffer is added into obtained precipitating, mixes, and 37 DEG C of incubation 30min are obtained
To the antibody-solutions of reduction, which is stored in the 50kDa centrifugal ultrafiltration pipe；
Wherein the preparation method of the TCEP-R-Buffer of every 1000 μ L is as follows: molten to 8 μ L 0.5M tri- (2- carboxyethyl) phosphines
It is added in 992 μ L R-Buffer, mixes in liquid (TCEP solution).
S3, purified metal load: to molecular cut off be 3kDa centrifugal ultrafiltration pipe in 200 μ L L-Buffer are added, then
The 100 μ L of metal load object solution that S1 is obtained is added, mixes, room temperature 12,000g is centrifuged 25min, continuously adds 300 μ L C-
Buffer is mixed, room temperature 12, and 000g is centrifuged 30min, and recycling precipitating, this is precipitated as the metal load object solution of purifying；
S4 purifies CD133 antibody: being added 300 into the antibody-solutions 50kDa centrifugal ultrafiltration pipe containing reduction that S2 is obtained
μ L C-Buffer, 12,000g room temperatures are centrifuged 10min, abandon supernatant, then add 400 μ L C-Buffer and mix, and 12,000g
Room temperature is centrifuged 10min, abandons supernatant, the precipitating of recycling, this is precipitated as the CD133 antibody of purifying.
S5 prepares CD133 antibody complex: the precipitating recycled in S3 is resuspended with 60 μ L C-Buffer, and re-suspension liquid is turned
It moves in the 50kDa centrifugal ultrafiltration pipe of the CD133 antibody equipped with purifying in S4, mix (will purify obtained in re-suspension liquid and S4
CD133 antibody mix), 37 DEG C of incubation 60min obtain CD133 antibody complex；
S6, clean CD133 antibody complex: with W-Buffer clean S5 obtained in CD133 antibody complex, done
Net CD133 mass spectrum streaming antibody, the specific steps are as follows: the 50kDa centrifugation containing CD133 antibody complex obtained to S5 is super
300 μ L W-Buffer are added in chimney filter, mix, 12,000g centrifugation 10min abandon supernatant, then washed with 400 μ L W-Buffer
Precipitating three times, is eventually adding 50 μ L W-buffer, rinses, and collects solution A in super filter tube；Then by centrifugal ultrafiltration pipe 1000g
It is centrifuged 2min, then rinses tube wall with 50 μ L W-buffer, solution B in super filter tube is collected, merges solution A and solution in super filter tube
B obtains clean CD133 mass spectrum streaming antibody, and the pure antibody of CD133 combines lanthanide series metal at this time, and 4 DEG C save backup.
S7 surveys the 280nm absorbance value of CD133 mass spectrum streaming antibody, CD133 mass spectrum streaming antibody concentration is calculated,
Returned to zero with W-Buffer, calculate the pure antibody of CD133 be converted into CD133 mass spectrum streaming antibody the rate of recovery be 60% or so；
CD133 mass spectrum streaming antibody is diluted to final concentration 0.5mg/mL, in antibodies buffer with commercially available antibodies buffer
Addition 0.05g/100ml Sodium azide in advance measures the potency of CD133 mass spectrum streaming antibody between 1:50 to 1:200.
Fig. 1 is that the CD133 mass spectrum streaming antibody (abbreviation 172Yb_CD133 (v) in figure) of the embodiment of the present invention does not set circle
The expression of door；Wherein Figure 1A is the whole expression for not setting circle door 172Yb_CD133 (v), and Figure 1B is TSNE1 multidimensional
The channel case generated after Data Dimensionality Reduction, Fig. 1 C are the channel cases generated after TSNE2 multidimensional data dimensionality reduction.
Fig. 2 is the CD133 mass spectrum streaming antibody (abbreviation 172Yb_CD133 (v) in figure) of the embodiment of the present invention in living cells
In expression；Wherein Fig. 2A is the whole expression of 172Yb_CD133 (v), after Fig. 2 B is TSNE1 multidimensional data dimensionality reduction
The channel case of generation, Fig. 2 C are the channel cases generated after TSNE2 multidimensional data dimensionality reduction.
Figure 1A, Figure 1B, Fig. 1 C, Fig. 2A, data are increasing to the right for abscissa in Fig. 2 B, Fig. 2 C, the upward data of ordinate
Also increasing, Figure 1A, Figure 1B, Fig. 1 C, Fig. 2A, the circle in Fig. 2 B, Fig. 2 C indicate the larger situation of data.It can be with from Fig. 1
Find out, it is mutual not set the channel case generated after the whole expression and multidimensional data dimensionality reduction of circle door 172Yb_CD133 (v)
Verifying, it was demonstrated that the whole expression for not setting circle door 172Yb_CD133 (v) is good, and expression quantity is high.Figure it is seen that
The channel case generated after whole expression of the 172Yb_CD133 (v) in living cells and multidimensional data dimensionality reduction is mutually authenticated,
Prove that the whole expression of 172Yb_CD133 (v) in living cells is good, expression quantity is high.
Fig. 3 is the background that the CD133 mass spectrum streaming antibody test of the embodiment of the present invention is；Wherein Fig. 3 A is single living thin
Background in born of the same parents' detection, Fig. 3 B are the Backgrounds in the detection of all living cells.As seen from Figure 3, in single living cell
And in all living cells detection process, the CD133 mass spectrum streaming that most cells can express the embodiment of the present invention is anti-
Body, and the basic crimping of negative cells of CD133 mass spectrum streaming antibody is not expressed, without peak value, utilize CD133 mass spectrum of the invention
The negative cells background that streaming antibody test goes out is very low, and it is very high to also illustrate that CD133 mass spectrum streaming antibody has in living cells
Expression intensity.
It should be noted that numberical range involved in method of the invention, it should be understood that in the numberical range
The solution of the present invention can be achieved in any value, since its bring effect is similar, therefore only retouches in the specific embodiment of the invention
Preferred embodiment 1 has been stated, although preferred implementation of the invention has been described, 1, those skilled in the art once learn
Basic creative concept, then additional changes and modifications may be made to these embodiments.So appended claims are intended to solve
It is interpreted as including preferred embodiment and all change and modification for falling into the scope of the invention.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
Mind and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to include these modifications and variations.
Claims (9)
1. a kind of CD133 mass spectrum streaming antibody, which is characterized in that the CD133 mass spectrum streaming antibody is with lanthanide series metal solution
It is raw material with the pure antibody of CD133, by what is be prepared after on lanthanide series metal load to the pure antibody of CD133.
2. CD133 mass spectrum streaming preparation method for antibody according to claim 1, which comprises the following steps:
S1 prepares metal load object solution: preparing lanthanide series metal solution, it is molten to obtain metal load object by 37 DEG C of incubation 30-40min
Liquid；
The reduction of S2, CD133 antibody: the pure antibody of R-Buffer and CD133 being added into molecular cut off 50kDa centrifugal ultrafiltration pipe,
It mixes, centrifugation abandons supernatant, TCEP-R-Buffer is added into obtained precipitating, mixes, and 37 DEG C of incubation 15-30min are gone back
Former antibody-solutions；
Wherein, the preparation method of the TCEP-R-Buffer of every 1000 μ L is as follows: 992 μ L are added into 8 μ L 0.5M TCEP solution
R-Buffer is mixed；
Purified metal load: S3 is added L-Buffer into molecular cut off 3kDa centrifugal ultrafiltration pipe, adds what S1 was obtained
Metal load object solution mixes, and centrifugation continuously adds C-Buffer, mixes, centrifugation, recycling precipitating；
S4 purifies CD133 antibody: C-Buffer is added in the antibody-solutions obtained to S2, is centrifuged, abandons supernatant, then adds
C-Buffer is mixed, centrifugation, abandons supernatant, the precipitating of recycling, this is precipitated as the CD133 antibody of purifying；
S5 prepares CD133 antibody complex: the precipitating recycled in S3 is resuspended with C-Buffer, and will obtain in re-suspension liquid and S4
The CD133 antibody of purifying mix, 37 DEG C of incubation 60-120min obtain CD133 antibody complex；
S6, clean CD133 antibody complex: with W-Buffer clean S5 obtained in CD133 antibody complex, obtain clean
CD133 mass spectrum streaming antibody；
Wherein the effect of R-Buffer is antibodies buffer；The effect of L-Buffer and C-Buffer is lanthanide series metal buffer；
The effect of W-Buffer is cleaning solution.
3. CD133 mass spectrum streaming preparation method for antibody according to claim 2, which is characterized in that in S1, the group of the lanthanides gold
Belong to the final concentration of 2.5mM of solution, the lanthanide series metal is ytterbium chloride or lanthanum chloride.
4. CD133 mass spectrum streaming preparation method for antibody according to claim 3, which is characterized in that in S2, R-Buffer:
The pure antibody of CD133: the volume ratio of TCEP-R-Buffer is 300:100-200:100.
5. CD133 mass spectrum streaming preparation method for antibody according to claim 4, which is characterized in that in S3, metal load object
The volume ratio of solution, L-Buffer and C-Buffer is 100:200:300.
6. CD133 mass spectrum streaming preparation method for antibody according to claim 5, which is characterized in that in S4, be added for the first time
C-Buffer, second C-Buffer and S2 being added in the volume ratio of TCEP-R-Buffer that is added be 300:
400-500:100.
7. CD133 mass spectrum streaming preparation method for antibody according to claim 2, which is characterized in that the specific steps of S6 are such as
Under: 300 μ L W-Buffer are added into the 50kDa centrifugal ultrafiltration pipe containing CD133 antibody complex that S5 is obtained, mix,
12,000g centrifugation 10min abandon supernatant, then wash precipitating three times with 400 μ L W-Buffer, are eventually adding 50 μ L W-buffer,
It rinses, collects solution A in super filter tube；Then the centrifugal ultrafiltration pipe 1000g is centrifuged 2min, then is rinsed with 50 μ L W-buffer
Tube wall collects solution B in super filter tube, merges solution A and solution B in super filter tube, obtains clean CD133 mass spectrum streaming antibody.
8. application of the CD133 mass spectrum streaming antibody according to claim 1 as mass spectrum flow cytometer detection stem cell markers.
9. the method according to claim 11 is preparing the application in mass spectrum flow cytometer detection stem cell markers.
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| CN109323908A (en) * | 2018-10-10 | 2019-02-12 | 浙江大学 | A kind of detection method for mass spectrum streaming technology |
| CN110412287A (en) * | 2019-07-11 | 2019-11-05 | 上海宸安生物科技有限公司 | One kind being based on single celled immunocyte parting quantitative analysis method |
| CN111982789B (en) * | 2020-08-21 | 2022-02-08 | 中国科学院生态环境研究中心 | High-throughput detection method of metal ions and metal nanoparticles based on single-cell enrichment and single-cell mass spectrometry |
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Also Published As
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| CN108398560A (en) | 2018-08-14 |
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