CN105950552A - Separation method for plasma cells capable of secreting antigen specific antibodies - Google Patents
Separation method for plasma cells capable of secreting antigen specific antibodies Download PDFInfo
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Abstract
The invention discloses a separation method for plasma cells capable of secreting antigen specific antibodies. The plasma cells capable of secreting the antigen specific antibodies are obtained through separating a plasma cell marker CD38 or CD138 mono-antibody and anti-IgG antibody cross-linked double-functional antibody and a magnetic bead which is crossly linked with a specific antigen. By adopting the separation method, the plasma cells capable of secreting the antigen specific antibodies can be accurately obtained, the working amount is extremely reduced, and the success rate is higher.
Description
Technical field
The invention belongs to monoclonal antibody technique field, be specifically related to a kind of plasmacytic point of secretion antigen specific antibody
A kind of method preparing antigenic specificity monoclonal antibody from method and high flux.
Background technology
Since first antibody in 1986 is applied to treatment, the most more than 50 antibody medicine is applied to clinical treatment,
About 70 antibody drugs will be had to enter clinical treatment application to the year two thousand twenty, it is contemplated that its market is up to 125,000,000,000 dollars.Add
The detection antibody of clinical diagnosis and the antibody of laboratory scientific research.The market of antibody is by the most about 125,000,000,000 dollars.Due to
Most monoclonal antibodies derives from mice, and mouse antibodies is foreign protein for people, has the highest immunogenicity, easily draws
Playing the immunoreation of human antimouse antibody, the immunoreation of human antimouse antibody hinders murine antibody significantly in clinical treatment
Application.It is primarily now to have two kinds of methods to solve this difficult problem.First is to carry out murine antibody by engineered method
Humanization.The antibody of the second preparation humanized.
Human antibody be mainly obtained by antibody hybridoma techniques, Phage displaying library technology and thin to people B
The approach of the conversion of born of the same parents.
(1) utilize human immunoglobulin gene transgenic mice as platform, by hybridoma antibody technology.At human immunoglobulin gene
Transgenic mice small mouse antibody gene is knocked, and therefore in human immunoglobulin gene transgenic mice, antibody is human antibody.
Hybridoma antibody technology was invented by British scientist Kother and Milstein in 1975.Miscellaneous from hybridoma antibody technology
Hand in 40 years since the invention of tumor antibody technique, be prepared for a large amount of monoclonal anti by authentic monoclonal antibody
Body, these monoclonal antibodies are widely used in scientific research, clinical diagnosis, clinical disease treatment.Hybridoma antibody technology
It is that the hybridoma B cell of secretion antigen specific antibody and the murine myeloma cell fusion of some drugs allergy formed is thin
Born of the same parents, this hybridoma not only has the function of B cell secretory antibody but also has the characteristic of myeloma cell's infinite multiplication, through sieving
Choosing can obtain the hybridoma cell strain of secretion antigen specific antibody.Utilize the production antibody that hybridoma cell strain can be unlimited.
(2) heavy chain of different antibodies and variable region of light chain are illustrated in the surface of phage by display technique of bacteriophage.Profit
Screening with target antigen, can obtain can be in conjunction with the antibody of target antigen.Utilize technique for gene engineering, by the heavy chain of the antibody of people and
The surface being illustrated in phage is cloned and expressed to chain variable region gene, sets up people's antibody phage display library.Utilize target
Antigen screens, people's antibody of available specific bond target antigen.The success or not in phage antibody display library determines
Source in the variable region sequences of antibody.The multiformity of sequence, and the multiformity of CDRs.Owing to cannot utilize target that people is entered
Row immunity.Be used for building the heavy chain of people's antibody of people's antibody phage display library and chain variable region gene mainly from without
The B cell clone of the people of immunity obtains.Because not through the affine maturation process of somatic mutation, this method is had to prepare
Antibody affine the lowest, specificity is the highest.And owing to the viscosity of phage is higher.Hold when screening antibodies specific antibody
Easily obtain false-positive bacterial strain.The particularly antibody of cell surface antigen.
(3) utilizing Epstein-Barr virus to convert human B cell is also a kind of approach obtaining human antibody.But it is thin that Epstein-Barr virus converts people B
The success rate of born of the same parents is the highest.And the B cell of mice is insensitive to Epstein-Barr virus.Although having invented virus-free transformation technology, but become
Power is more much lower than hybridoma technology.
Therefore obtain the specific antibody of high-affinity or how to be prepared by hybrid antibody technic.When with the albumen of purification
When matter antigen or other mice immunized with antigen, antigen activates T cell and the B cell of mice through processing submission, in the ginseng of T cell
Under with, B cell is through propagation, differentiation, and the affine maturation process of somatic mutation ultimately becomes can secretion antigen specific antibody
B cell (plasma cell).Plasma cell is the ultimate cell of B cell differentiation.Plasma cell surface not antigen expressed receptor.Plasma cell is
Affine maturation process through somatic mutation.Therefore the affinity of antibody of plasma cell secretion is the highest.Plasma cell divides
It is melted into ripe rear entrance blood and bone marrow.Owing to plasma cell life cycle is shorter, the plasma cell in blood can continuous apoptosis, from you
Make the lowering of concentration of antibodies in blood.But the plasma cell in bone marrow, has part to survive, and these cells constantly produce
Raw antibody, to maintain the concentration of antibody in blood plasma.
Although hybridoma antibody technology is just invention as far back as 1975, but over 40 years, hybridoma antibody technology is the most great
Development and change.The lowest problem of formation rate of hybridoma does not the most solve.With mice during the proteantigen immune mouse of purification
10 can be produced5-106The B cell of antigenic specificity, the antibody of the B cell secretion of these antigenic specificities identifies on antigen not respectively
Same antigenic determinant, for each antigenic determinant, the quantity of antibody is different, and some is that main antigen determines
Bunch, therefore these main antigenic determinants activate the B cell of the antibody forming more these epitopic specificity of secretion.
But success rate is low during owing to utilizing hybridoma technology to prepare antibody, can form 100 after a general fusion can secretory antibodies
Hybridoma, this most only account for total secretion antigen-specific antibodies B cell 0.1%.Obtain more the most more quickly and effectively
The exploitation accelerating antibody drug is had very important significance by the antibody of the different loci of combining target proteantigen.Antibody is controlled
Mostly the target site treated is protein (protein receptor, protein receptor binding thing and enzyme), and each protein has multiple antigen position
Point, the site of antibody binding proteins antigen is different, and its effect is also the most different, the function of some site energy Activin receptor, has
The function of protein receptor can be suppressed a bit.People can be according to pathogenic mechanism, and the approach for the treatment of takes the desired antibody of selection.
Chinese patent application 201110119289.5 discloses a kind of method for preparing monoclonal antibody, uses thin to lymph B
Born of the same parents cultivate the magnetic particulate microsphere adding immobilization antigen in micropore, and magnetic separation magnetic particulate microsphere is to corresponding microwell plate, immunization
Learn luminescence method or immunofluorescence method detects the single B cell secreting specific antibody, by single-cell RT-PCR and nest-type PRC
Method amplify that antibody is light, heavy chain variable region gene recombinating to the expression plasmid comprising antibody constant region, transfecting host is thin
Cellular expression monoclonal antibody.The method first carries out identifying positive colony, the most again variable region of clonal antibody from positive colony
Gene, due to the enormous amount of B cell, screening operation is the most difficult.Simultaneously because detection and the separation of clone, easily cause B
The loss of cell.
Chinese patent application CN201280016777.6 discloses a kind of plasma cell and prepares the technology of antibody.This technology should
With plasmacytic fluorochrome label plasma cell.Then all of plasma cell is separated, then carry out gene clone, due to anti-
It is relatively low that the plasma cell of former specific antibody accounts for total plasma cell ratio, and therefore specificity is poor, and efficiency comparison is low.This skill
Art ratio is relatively time-consuming, and workload is big, needs substantial amounts of screen plate, has manually been difficult to such screening, it is therefore necessary to full-automatic
Machine complete screening.
Summary of the invention
The present invention is directed to prior art not enough, utilize individual cells antibody cloning technology, antibody expression technology and list
The high-throughout high-affinity antibody preparing antigenic specificity from plasma cell of cell separation culture technique.
The concrete technical scheme of the present invention
A kind of plasmacytic separation method of secretion antigen specific antibody, comprises the steps:
(1) after using specific antigen immunity inoculation human or animal, it is thus achieved that after lymphocyte, isolated plasma cell；
(2) in plasma cell, add the monoclonal antibody of plasma cell Specific marker and the bifunctional antibody of anti-igg antibody crosslinking, thus
Plasma cell surface formed the affine layer of anti-igg antibody, when cultivating in vitro, the IgG antibody of plasma cell secretion once secretion just by
Catch at cell surface；
(3) add and be crosslinked with the magnetic bead of specific antigen, formed plasma cell-bifunctional antibody complex-IgG antibody-specific antigen-
Bead complexes；
(4) plasma cell-bifunctional antibody complex-IgG antibody-specific antigen-bead complexes is carried out Magnetic Isolation, obtain
The plasma cell of secretion antigen specific antibody.
Above-mentioned specific antigen refers to obtain the antigen used by purpose antibody.
After described lymphocyte refers to animal or people's immunity, gather from lymph fluid, lymphoid tissue, blood cell samples or bone
Marrow.
Plasmacytic separation method is this area conventional method, and available plasma cell separating kit completes (CD138+
Plasma Cell Isolation Kit, mouse, Plasma Cell Isolation Kit II, human Miltenyl
Biotec).
Above-mentioned plasma cell Specific marker is CD38 or CD138.When preferably object of inoculation is behaved, plasma cell specificity mark
When will thing selects CD38, preferably object of inoculation to be Mus, plasma cell Specific marker selects CD138.
CD38 and CD138 is the Specific marker on plasma cell surface, and CD38 antibody can be with plasma cell surface of cell membrane
CD38 specific binding；CD138 antibody can be specific binding with the CD138 of plasma cell surface of cell membrane, anti-igg antibody energy
Enough antibody specificities in animal body are combined.
The antibody that the monoclonal antibody of described plasma cell Specific marker cross-links with anti-igg antibody, as CD38 monoclonal antibody-anti-igg is double
Function antibody or CD138 monoclonal antibody-anti-igg bifunctional antibody can be prepared by cross-linking agent by chemical crosslink technique.
This area routine techniques preparation can be used to be crosslinked with the magnetic bead of specific antigen.
Another object of the present invention is a kind of method providing high flux to prepare antigenic specificity monoclonal antibody, in employing
Described method prepares the plasma cell of secretion antigen specific antibody, utilizes singe-cell PCR gene clone technology clone each
Plasmacytic heavy chain of antibody and chain variable region gene, by gene order and the analysis of aminoacid sequence, debate different with rice steamer
Antibody.To there is the antibody cloning of different aminoacids sequence in expression vector.Then restructuring is to specific prokaryotic expression
In carrier, then convert escherichia coli and express, utilize target antigen to screen expression product, available specific bond mesh
The antibody library of mark antigen.
Another object of the present invention is to provide a kind of for separating the plasmacytic test kit of secretion antigen specific antibody, its
It is characterised by including CD38 or CD138 monoclonal antibody and the bifunctional antibody of anti-igg antibody crosslinking and being crosslinked with the magnetic bead of specific antigen.
Another object of the present invention is the test kit providing a kind of high flux to prepare antigenic specificity monoclonal antibody, and it is special
Levy and be to include CD38 or CD138 monoclonal antibody and the bifunctional antibody of anti-igg antibody crosslinking, be crosslinked with the magnetic bead of specific antigen, DNA
Extractor system, PCR amplification system and expression system.
The present invention utilizes plasma cell isolation technics and genetic engineering antibody technology to be combined a kind of of foundation and produces high parent
With the method resisting body strenuously.First plasma cell is separated from blood or in bone marrow, more special from plasma cell separation secretion antigen
The plasma cell of heterogenetic antibody.The plasma cell of secretion antigen specific antibody is separated in aperture, forms a cell each little
Hole, utilizes singe-cell PCR gene clone technology, the V of clonal antibodyH、VLGene.These antibody genes are entered in escherichia coli
Row is expressed.This method specificity is high, can efficiently prepare the specific antibody of substantial amounts of conjugated antigen different loci.This method is with existing
There is technology to compare, it is possible to first to be separated by specific for secretion antigen antibody plasma cell, then carry out gene clone, and existing skill
Being to be separated by all of plasma cell under art, therefore the present invention is with clearly defined objective, it is possible to accurately obtain secretion antigen specificity
Antibody plasma cell, considerably reduce workload, success rate is higher.
Detailed description of the invention
As a example by using human albumin as antigen, technical solution of the present invention is described.
The plasmacytic preparation of embodiment 1
Freund's complete adjuvant is mixed and made into Emulsion with human albumin solution in the ratio of 1:1.Lumbar injection immunity 6-8 week old is female
Property Balb/c mice (every mice 100 microlitre, containing 10 microgram human albumins).With incomplete Freund's adjuvant lumbar injection after two weeks
10 microgram human albumin booster immunization mices.The titer of hematometry antibody is taken after two weeks.Through the tail vein injection 10 white egg of microgram people
White booster immunization mice, after four days, by sacrifice, separating spleen cell.Utilize plasma cell separating kit (CD138+
Plasma Cell Isolation Kit, mouse, Miltenyl Biotec) from spleen cell, separate plasma cell.
The preparation of embodiment 2 bifunctional antibody
CD138 monoclonal antibody and mouse-anti IgG monoclonal antibody covalent cross-linking are prepared difunctional by the method using chemical crosslinking
Antibody.With BS3 (Thermo Scientific, Cat. #21580), as cross-linking agent, related experiment is with reference to the operation of this reagent
Explanation is carried out.Antibody affinity column after crosslinking separates.
Embodiment 3 antigen and the crosslinking of magnetic bead
Human albumin is cross-linked by method and the magnetic bead of chemical crosslinking.Prepare antigen magnetic bead cross-linking agent, divide for separating
Secrete the plasma cell of human albumin specific antibody.Human albumin is dissolved in reaction buffer (0.05 M MES, 0.5 M
NaCl, pH 6.0) in so that final concentration of 1mg/ml.Magnetic bead surfaces is activated with final concentration of 2mM EDC and 5mM sulfo group-NHS
Carboxyl.Will activation after magnetic bead mix with antigen, under room temperature shake hatch 2 hours.Under magnetic field, magnetic bead is precipitated,
Abandon supernatant, wash magnetic bead 2 times with buffer.
Embodiment 4 secretion antigen specific antibody plasma cell separates
Prepare to embodiment 1 method and plasma cell adds the CD138-mouse-anti IgG bifunctional antibody that embodiment 2 prepares, be used for marking
Note plasma cell, on plasma cell cell membrane, CD138 and CD138-mouse-anti IgG bifunctional antibody combines, and forms one on plasma cell surface
The affine layer of individual mouse-anti IgG antibody, cell cultivates certain time in vitro, and the antibody of plasma cell secretion is just captured once secrete
On plasma cell surface, it is eventually adding the human albumin magnetic bead cross-linking products that embodiment 3 prepares, reverse hybrid reaction on shaking table
15min, human albumin and the antibodies caught on plasma cell surface, carry out Magnetic Isolation subsequently, abandon supernatant, and secretion antigen is special
The plasma cell of heterogenetic antibody is present in precipitation.Cell suspension is made single cell suspension.
Embodiment 5 utilizes singe-cell PCR technology clonal antibody VH、VLGene
The plasma cell of secretion human albumin specific antibody separation obtained is by the aperture of cell transposition 384 orifice plate, each
Cell per well, utilizes singe-cell PCR technology clonal antibody VH、VLGene.Primer used is as follows.
VH 5 ' holds primer:
(1) GGGAATTCATGRASTTSKGGYTMARCTKGRTTT (SEQ ID No:1)；
(2) GGGAATTCATGRAATGSASCTGGGTYWTYCTCTT (SEQ ID No:2)；
(3) ACTAGTCGACATGGACTCCAGGCTCAATTTAGTTTTCCT (SEQ ID No:3)；
(4) ACTAGTCGACATGAAATGCAGCTGGRTYATSTTCTT (SEQ ID No:4)；
(5) ACTAGTCGACATGGGATGGAGCTRTATCATSYTCTT (SEQ ID No:5)；
(6) ACTAGTCGACATGAACTTYGGGYTSAGMTTGRTTT (SEQ ID No:6)；
VH 3 ' holds primer: CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG (SEQ ID No:7).
VL5 ' holds primer:
(1) GGGAATTCATGRAGWCACAKWCYCAGGTCTTT (SEQ ID No:8)；
(2) GGGAATTCATGGAGACAGACACACTCCTGCTAT (SEQ ID No:9)；
(3) ACTAGTCGACATGGAGWCAGACACACTSCTGYTATGGGT (SEQ ID No:10)；
(4) ACTAGTCGACATGAGGRCCCCTGCTCAGWTTYTTGGIWTCTT (SEQ ID No:11)；
(5) ACTAGTCGACATGAGTGTGCYCACTCAGGTCCTGGSGTT (SEQ ID No:12)；
(6) ACTAGTCGACATGAGIMMKTCIMTTCAITTCYTGGG (SEQ ID No:13)；
(7) ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT (SEQ ID No:14).
VL3 ' holds primer
(1) CCCAAGCTTACTGGATGGTGGGAAGATGGA (SEQ ID No:15)；
(2) CCCAAGCTTAGCTCYTCWGWGGAIGGYGGRAA (SEQ ID No:16).
PCR condition is as follows:
36.25 μ l deionized water
5 μl 10X NovaTaqPCR buffer
1 μ l dNTPs (final concentration 0.2 mM)
2.5 μ l 5'lea primers (10 pmol/ μ l)
2.5 μ l 3'lea primers (10 pmol/ μ l)
1 μl (1.25 U) NovaTaqDNA Polymerase
5 μl cDNA
Denature 1 min at 94°C
Anneal 1 min at 50°C
Extend 2 min at 72°C
Final Extension 6 min at 72°C
The expression of embodiment 6 antibody
IgG antibody VH obtained by gene clone, the gene of VL variable region checks order, by the amino to VH, VL variable region
Sequence analysis judges the similarities and differences of antibody.The most different antibody has different VH, the aminoacid sequence of VL variable region.
Variable region VH, the VL gene with different aminoacids sequence is cloned in expression vector, the expression vector of gained is transformed into
Escherichia coli carry out antibody-secreting expression, after isolated and purified antibody, for the detection of next step antibody characteristic.
The screening of embodiment 7 positive colony
Positive colony Screening to use ELISA method.Briefly, anti human albumin is coated elisa plate, 1 micrograms per millilitre, 4 degree
Overnight.Washing plate 3 times by washing liquid, added by confining liquid in aperture, room temperature is closed 2 hours.Outwell confining liquid, add test antibodies,
Incubated at room 2 hours.Washing plate 5 times by washing liquid, the anti-igg two adding alkali phosphatase enzyme mark resists, incubated at room 1 hour.With washing
Liquid washes plate 5 times, adds substrate and develops the color, and microplate reader reads OD410.
The preparation of anti human albumin's monoclonal antibody under embodiment 8 prior art
Mouse spleen prepared by method separation embodiment 1 method with reference to disclosed in Chinese patent application CN201280016777.6 is thin
The plasma cell of secreting specificity antibody in born of the same parents.The method using embodiment 5-7, utilizes singe-cell PCR gene clone technology, from slurry
Clonal antibody VH in cell, VL gene.Through gene order and amino acid sequence analysis, the most homotactic gene is cloned into table
Reach the expression carrying out antibody in carrier, utilize ELISA screening positive clone.
The result of the high-affinity antibody separating plasma cell and antigenic specificity with embodiment of the present invention 2-7 method contrasts,
Result is as shown in table 1.
Result shows the efficiency comparison height utilizing the method for the present invention to prepare monoclonal antibody, and success rate is bigger.It is easier to
Obtain the monoclonal antibody combining different loci.
SEQUENCE LISTING
<110>Ningbo Ai Ke bio tech ltd
<120>a kind of plasmacytic separation method of secretion antigen specific antibody
<130>
<160> 16
<210> 1
<211> 33
<212> DNA
<213>artificial sequence
<400> 1
GGGAATTCATGRASTTSKGGYTMARCTKGRTTT
<210> 2
<211> 34
<212> DNA
<213>artificial sequence
<400> 2
GGGAATTCATGRAATGSASCTGGGTYWTYCTCTT
<210> 3
<211> 39
<212> DNA
<213>artificial sequence
<400> 3
ACTAGTCGACATGGACTCCAGGCTCAATTTAGTTTTCCT
<210> 4
<211> 36
<212> DNA
<213>artificial sequence
<400> 4
ACTAGTCGACATGAAATGCAGCTGGRTYATSTTCTT
<210> 5
<211> 36
<212> DNA
<213>artificial sequence
<400> 5
ACTAGTCGACATGGGATGGAGCTRTATCATSYTCTT
<210> 6
<211> 35
<212> DNA
<213>artificial sequence
<400> 6
ACTAGTCGACATGAACTTYGGGYTSAGMTTGRTTT
<210> 7
<211> 35
<212> DNA
<213>artificial sequence
<400> 7
CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG
<210> 8
<211> 32
<212> DNA
<213>artificial sequence
<400> 8
GGGAATTCATGRAGWCACAKWCYCAGGTCTTT
<210> 9
<211> 33
<212> DNA
<213>artificial sequence
<400> 9
GGGAATTCATGGAGACAGACACACTCCTGCTAT
<210> 10
<211> 39
<212> DNA
<213>artificial sequence
<400> 10
ACTAGTCGACATGGAGWCAGACACACTSCTGYTATGGGT
<210> 11
<211> 42
<212> DNA
<213>artificial sequence
<400> 11
ACTAGTCGACATGAGGRCCCCTGCTCAGWTTYTTGGIWTCTT
<210> 12
<211> 39
<212> DNA
<213>artificial sequence
<400> 12
ACTAGTCGACATGAGTGTGCYCACTCAGGTCCTGGSGTT
<210> 13
<211> 36
<212> DNA
<213>artificial sequence
<400> 13
ACTAGTCGACATGAGIMMKTCIMTTCAITTCYTGGG
<210> 14
<211> 39
<212> DNA
<213>artificial sequence
<400> 14
ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT
<210> 15
<211> 30
<212> DNA
<213>artificial sequence
<400> 15
CCCAAGCTTACTGGATGGTGGGAAGATGGA
<210> 16
<211> 32
<212> DNA
<213>artificial sequence
<400> 16
CCCAAGCTTAGCTCYTCWGWGGAIGGYGGRAA
Claims (5)
1. the plasmacytic separation method of secretion antigen specific antibody, it is characterised in that comprise the steps:
(1), after using specific antigen immunity inoculation human or animal, it is thus achieved that after lymphocyte, from lymphocyte, isolated slurry is thin
Born of the same parents；
(2) in plasma cell, the monoclonal antibody of plasma cell Specific marker and the bifunctional antibody of anti-igg antibody crosslinking are added, at slurry
Cell surface forms the affine layer of anti-igg antibody, and when cultivating in vitro, the IgG antibody of plasma cell secretion is just captured once secretion
At cell surface；
(3) add and be crosslinked with the magnetic bead of specific antigen, formed plasma cell-bifunctional antibody complex-IgG antibody-specific antigen-
Bead complexes；
(4) plasma cell-bifunctional antibody complex-IgG antibody-specific antigen-bead complexes is carried out Magnetic Isolation, obtain
The plasma cell of secretion antigen specific antibody.
2. separation method as claimed in claim 1, it is characterised in that described plasma cell Specific marker be CD38 or
CD138。
3. the method that a high flux prepares antigenic specificity monoclonal antibody, it is characterised in that use described in claim 1
Method prepares the plasma cell of secretion antigen specific antibody, utilizes Antibody library, clones each plasma cell repertoire antibody
Heavy chain and chain variable region gene, then recombinate in specific prokaryotic expression carrier, then convert escherichia coli and express, right
Expression product utilizes specific antigen to screen, the antibody library of available specific bond specific antigen.
4. one kind is used for separating the plasmacytic test kit of secretion antigen specific antibody, it is characterised in that include CD38 or CD138
The bifunctional antibody of monoclonal antibody and anti-igg antibody crosslinking and be crosslinked with the magnetic bead of specific antigen.
5. a high flux prepares the test kit of antigenic specificity monoclonal antibody, it is characterised in that include that CD38 or CD138 is mono-
Anti-bifunctional antibody with anti-igg antibody crosslinking, be crosslinked with the magnetic bead of specific antigen, DNA extractor system, PCR amplification system and
Expression system.
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| CN201610356287.0A CN105950552A (en) | 2016-05-26 | 2016-05-26 | Separation method for plasma cells capable of secreting antigen specific antibodies |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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