CN108342364A - A kind of SPA mediates the hybridoma technology of double antibody capture - Google Patents
A kind of SPA mediates the hybridoma technology of double antibody capture Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
Abstract
A kind of SPA for biomedical sector mediates the hybridoma technology of double antibody capture,It is characterized in that by myeloma cell (Ag1) and anti-myeloma cell antibody (Ab1),B cell (Ag2) and anti-B cell antibody (Ab2) press 1: 1 mixing respectively,Make that immune combine respectively occurs,It is respectively formed the conjugate of Ag1 Ab1 and Ag2 Ab2,Again by two kinds of conjugates by 1: 1 mixing,IgGFc sections of the half of Ab1 is combined with the Fc receptors of Ag2 in Ag2 Ab2 conjugates in Ag1 Ab1 conjugates at this time,Form the conjugate of Ag1 Ab1 Ag2 Ab2,Half Ag1 is set to be connected to Ag2 by Ab1,Staphylococcal protein A (SPA) is added at this time,By the IgGFc receptors of SPA,It is separately connected the IgGFc not being combined in IgGFc the and Ag2 Ab2 not being combined in Ag1 Ab1,To in SPA,Under the action of Ab1 and Ab2,Specificity,The distance for myeloma cell and the B cell of furthering proportionally,In concanavalin A,Under the further effect of IFN γ and low concentration PEG,It is easy to the contact and fusion of after birth.
Description
Technical field
The present invention relates to the hybridoma technologies that a kind of SPA of biomedical sector mediates double antibody capture.
Background technology
Hybridoma technology, that is, hybridoma technology, also known as monoclonal antibody technique.
Ke Le (Kohler) and Millstein (Milstein) (1975) prove that myeloma cell and immune animal spleen are thin
Born of the same parents hybridize, and form antibody -- the monoclonal antibody for the high specific that can secrete the homogeneous for the antigen, and this technology is commonly referred to as
Hybridoma technology.Myeloma cell in vitro can be with continuous passage, and splenocyte is thesocyte, cannot be bred in vitro.Such as
The myeloma cell of mouse is hybridized with the lymphocyte for secreting certain antibody or the factor, then hybrid cell both has tumour cell
The characteristic of Immortalization, and the ability with lymphocyte energy secreting specificity antibody or the factor, while also overcoming immune leaching
The shortcomings that bar cell cannot be bred in vitro, the cell of hybridization is known as hybridoma.
The purpose for establishing hybridoma technology is the monoclonal antibody prepared to antigen-specific, so one side of fused cell is necessary
The B cell that selection is immunized by antigen, is typically derived from the splenocyte of immune animal.Spleen is the important place of B cell aggregation, nothing
It is stimulated by with which kind of immunization ways, apparent antibody response reaction is all will appear in spleen.Another party of hybrid cell be then in order to
The continuous proliferation of cell, only tumour cell just have this characteristic after holding cell hydridization.Select the cell of same system can
Increase the success rate of hybridization.Huppert's disease is B cell system malignant tumour, so being ideal splenocyte hybridization companion.
Hybridoma technology grows up on the basis of cell fusion, so hybridoma technology is also referred to as cell fusion
Technology, cell fusion are a random physical processes.In mouse boosting cell and murine myeloma cell mixed cell suspension
In, cell will occur in a variety of forms after hybridization.As the splenocyte and oncocyte of fusion, the splenocyte of fusion and splenocyte,
The multimeric forms etc. of the oncocyte and oncocyte of fusion, the splenocyte not merged, the oncocyte and cell that do not merge.Normally
Splenocyte in the medium only survive 5~7d, without especially screening；The multimeric forms of cell are also easy to die；And do not melt
The oncocyte of conjunction then needs to carry out particularly screening removal.
In view of two approach of cell DNA synthesis, main path therein is by sugar and Amino acid synthesis nucleotide, in turn
Synthetic DNA, folic acid participate in this building-up process as important coenzyme.Another accessory pathway is in hypoxanthine and thymidine
In the presence of nucleosides, the catalysis through Hypoxanthine ribose phosphate invertase (HGPRT) and thymidine kinase (TK)
Act on synthetic DNA.There are 3 kinds of key components in the Selective agar medium of cell fusion：Hypoxanthine (hypoxanthine, H), first ammonia
Pterin (aminopterin, A) and thymidine (thymidine, T), so the prefix of three is taken to be known as HAT culture mediums.
Methotrexate (MTX) is the antagonist of folic acid, and oncocyte can be blocked to utilize usual channel synthetic DNA, and merge oncocyte used be through
The HGPRT- cell strains that toxicity culture medium is selected, so cannot be grown in the culture medium.Only fused cell has parental generation double
The heritability of side, can be in HAT culture mediums long-term survival and breeding.So HAT culture mediums selection splenocyte and tumor can be used thin
The effective integration of born of the same parents removes the invalid splenocyte for merging and not merging, not of splenocyte and splenocyte, oncocyte and oncocyte
The invalid fusion of the forms such as the oncocyte of fusion, the polymer of cell.
Currently, hybridoma technology still continues to use Ke Le (Kohler) and Millstein (Milstein) (1975) wound
The myeloma cell built and immune animal splenocyte hybridoma technology (PEG methods), the technology are established on the basis of cell fusion,
Cell fusion agent used is polyethylene glycol (PEG1000~2000), although PEG is still presently preferred fusion agent, PEG pairs
The toxic effect of cell is very big, and concentration more high toxicity is bigger, effective integration dosage (50%~80%) range PEG still to thin
Born of the same parents have prodigious toxicity, this greatly affected the cell survival rate after effective cell fusion rate and fusion.
Invention content
To solve the above-mentioned problems, a kind of low PEG dosage, hypotoxicity, SPA mediation double antibody captures hybridization is established
Tumor technology, it is proposed that the present invention.
The invention aims to provide a kind of hybridoma technology that SPA mediates double antibody to capture.
The object of the present invention is achieved like this：Myeloma cell (Ag1) and anti-myeloma cell antibody (Ab1), B is thin
Born of the same parents (Ag2) and anti-B cell antibody (Ab2) press 1: 1 mixing respectively, make respectively to occur it is immune combine, be respectively formed Ag1-Ab1 and
The conjugate of Ag2-Ab2, then by two kinds of conjugates by 1: 1 mixing, at this time in Ag1-Ab1 conjugates IgGFc section of the half of Ab1 and
The Fc receptors of Ag2 combine in Ag2-Ab2 conjugates, form the conjugate of Ag1-Ab1-Ag2-Ab2, half Ag1 is made to be connected by Ab1
To Ag2, staphylococcal protein A (SPA) is added at this time and is separately connected in Ag1-Ab1 not by the IgGFc receptors of SPA
The IgGFc not being combined in the IgGFc and Ag2-Ab2 that are combined, to which under the action of SPA, Ab1 and Ab2, specificity has
The distance for myeloma cell and the B cell of furthering to ratio, in the further effect of concanavalin A, IFN-γ and low concentration PEG
Under, it is easy to the contact and fusion of after birth.
Present invention discover that SPA expression IgGFc receptors, can combine IgGFc sections, B cell more than half also express IgGFc by
Body can also combine IgGFc sections, and IFN-γ can activate IgGFc receptors, cell fusion, concanavalin A is promoted cell to be inhibited to transport
Dynamic, promotion cell agglutination and extension time-to-live, pre-confluent agent is designed accordingly, handles cell to be fused, mediated through SPA and dual anti-
Body captures, and makes B cell to be fused and myeloma cell specific bond pari passu, forms the anti-B cell antibody-SPA- of B cell-
The conjugate of anti-myeloma cell antibody-myeloma cell, further cell distance, is easy to after birth contact, and then in certain concentration
IFN-γ, concanavalin A and under the action of the fusion agent (PEG) of hypotoxicity, make to be fused cell-specific due to low concentration
Fusion, reduces invalid fusion, increases the effective integration of B cell and myeloma cell and the survival rate of passage cell.
Specific implementation mode
Fig. 1 is that the SPA of the present invention mediates double antibody capture to help and melt schematic diagram.
Fig. 2 is the cell fusion figure of the present invention.
In Fig. 1,1 the staphylococcal protein A (SPA) with IgGFc receptors is indicated, 2 indicate that number is 6 and 7
Cell to be fused monoclonal antibody, 3 indicate number be 4 and 5 cell to be fused monoclonal antibody, in monoclonal antibody
[2] after the cell combination to be fused that the cell combination to be fused for being 6 and 7 with number, monoclonal antibody [3] are 4 and 5 with number,
Connection through SPA again makes the cell that number is 4,5,6 and 7 be conducive to merge under the action of PEG.
In fig. 2, it under the helping and melt of monoclonal antibody and/or SPA, screens 2 weeks through HAT, is shot under inverted microscope
(40X), obtains hybridoma cell clone.
With reference to Fig. 1 and Fig. 2, embodiment of the present invention is described in detail.
1, experiment reagent
(1) pre-confluent agent：1. basal liquid：A concentration of 4.6 μ g/L IFN-γ, 2.5 μ g/ml are incorporated in DMEM culture mediums
Concanavalin A；2. solution A：Antibody (the B cell: antibody=1: 1) of the potency such as B cell is incorporated and is fused in basal liquid；③
Second liquid：Antibody (the myeloma cell: antibody=1: 1) of the potency such as myeloma cell is incorporated and is fused in basal liquid；4. third
Liquid：SPA (B cell or the myeloma of the potency such as B cell or myeloma cell or corresponding antibodies are incorporated and are fused in basal liquid
Cell or corresponding antibodies: SPA=1: 1).Wherein IFN-γ can activate IgGFc receptors, promote cell fusion；Concanavalin A can inhibit
Cell movement promotes cell agglutination and extends the time-to-live；Antibody, which can connect B cell and myeloma cell, SPA, can connect antibody
Fc sections, formed the anti-B cell antibody-SPA- anti-myeloma cells antibody-myeloma cell of B cell-conjugate, further cell
Distance is easy to after birth contact, and then in 4.6 μ g/LIFN- γ, the work of 2.5 μ g/ml concanavalin As and 25% fusion agent (PEG)
Under, make to be fused cell-specific fusion.
(2) cultivate reagent：DMEM culture mediums, HAT Selective agar mediums are purchased from Sigma companies, top grade fetal calf serum (FBS) purchase
The oceans Jinshi City Hao on daytime biological products science and technology responsibility Co., Ltd；DMSO (-- methyl sulfoxide) it is domestic analytical reagents；
2, cell fusion method
(1) preparation of myeloma cell：Fusion the last week takes out the myeloma cell (SP2/ that a pipe freezes out of liquid nitrogen container
0), be immediately placed in hot water thaw (using it is most be Sp2/0 cell strains, the cell strain growth and fusion efficiencies are good, itself is not
Any heavy chain immunoglobulin or light chain are secreted, the highest growth scale of cell is 9 × 105/ ml, the doubling time is usually 10~
15h；With selection homologous cell strain is considered in the relevant practical application of human body, if the Shanghai bio tech ltd Fu Xiang is to ATCC
The NCI-H929 human myeloma cells strain that cell bank is introduced).Appropriate complete culture solution is added after thawing, 1000r/m centrifuges 3min；
It is repeated 1 times.Sediment is moved into Tissue Culture Flask, adds DMEM culture solutions, sets CO2 incubator cultures, once passed within 3-4 days
In generation, expands culture, merges and adjusts cell state in first 24 hours, ensures that cellular morphology is good, it is vigorous to grow before fusion.Fusion
When SP2/0 is blown down from culture bottle, be transferred in centrifuge tube, 1000r/m centrifuges 5-10min, repeated washing cell 2 times, gently
Mixing is beaten, takes a small amount of suspension to count, adjusts density, empirically ensures that its density is 80% or so in fusion.
(2) preparation of bone-marrow-derived lymphocyte：B cell adjusts total cell number to 1 × 10 with DMEM culture solutions (basal medium)8
~2 × 108, it is used for cell fusion, expects that blue dyeing, phase-contrast microscopy, viable count should be higher than that 80% as qualification with platform.
(3) pre-confluent is handled：B cell and myeloma cell are separately added into first centrifuge tube and second with 10: 1-5: 1 ratio
In centrifuge tube, 1000r/min centrifuge 5min, discard supernatant, gently beat tube bottom to cell grainless precipitate, then first from
Heart Guan Zhongjia 2ml solution As, in second centrifuge tube plus 2ml second liquid, 37 DEG C 1 hour, by the suspension mixing of first, second centrifuge tube, 37 DEG C 1
Hour, add the third liquid, 37 DEG C 1 hour, 1000r/min centrifuges 5min, discards supernatant, gently beats tube bottom to cell without particle
Shape precipitates.
(4) cell fusion：Gently rotation preheats centrifuge tube in 37 DEG C of water-baths, by preheating under aseptic condition after taking-up
The 25%PEG3000 of 1000 μ L is added drop-wise to along tube wall in fusion pipe in 60s, while gently rotating centrifugal pipe, later will preheating
25mL basal mediums be also added drop-wise in centrifuge tube along tube wall in 3-5min, lightly rotating centrifugal during addition
Pipe, is then allowed to stand in 37 DEG C of water-bath 10min, and (remove combine antibody) is diffused antibody 5 minutes in 56 DEG C of water-baths of transposition, 1500r/m
Centrifuge 5min, discard supernatant, 50mL HAT culture mediums are added, be inoculated into 96 well culture plates after appropriate mixing, be placed in 37 DEG C,
It is cultivated in 5%CO2 incubators.
(5) screening of fused cell：96 orifice plate cell growth status are observed, only having effective integration cell after 7-10 days can
Growth discards HAT culture mediums at this time, replaces complete medium.When cell clone growth area reaches 1/10 cell hole, go to train
Support supernatant, selection has the culture hole of the good hybridoma cell strain of growth conditions, the position of label cell strain growth under microscope,
Size, using sterile pipette tips mark position draw cell clone to newly have in the culture hole of complete medium, then according to
Secondary doubling dilution to hole is counted below, and 37 DEG C, the interior culture of 5%CO2 incubators about 1 week, microscopically observation cell growth status waits for
When cell clone is covered with to 1/10 or more hole floor space, the characteristic of cell or culture supernatant detection fusion cell is taken.
(6) cell fusion effect compares：
(A) conventional chemical agent revulsion (PEG methods) and the pre-confluent rate (fusion rate before HAT selection cultures) of the present invention are right
Than：During cell fusion, other than B cell can be merged with myeloma cell, it also occur that B cell and B cell,
Myeloma cell is merged with myeloma cell's, and only B cell and myeloma cell is merged, and could to obtain both have B cell
Generating antibody characteristic again has the B cell hybridoma cell of myeloma cell's infinite multiplication characteristic, is only effective fusion, and B
Cell is all to merge in vain with B cell, myeloma cell and merging for myeloma cell.In order to compare conventional chemical agent revulsion
With the present invention pre-confluent rate, take respectively fusion after but HAT culture before cell precipitation film-making, dyeing, microscopic observation, count
100 cells calculate double-core or multi-nucleus cell number/total cell number, are converted into percentage (table 1), and wherein conventional chemical agent induces
The pre-confluent rate of method and the present invention is respectively 43%, 48%, and pre-confluent rate of the invention is higher than conventional chemical agent revulsion, but nothing
Significant difference (p ＞ 0.05).
(B) conventional chemical agent revulsion and the effective integration rate of the present invention compare：After HAT selections culture 10 days, only B
The hybridoma that cell merges (effective integration) with myeloma cell could stablize growth, and B cell and B cell, myeloma
The cell for merging (invalid fusion) and do not merge of cell and myeloma cell, cannot grow in HAT Selective agar mediums.
In order to compare the effective integration rate of the two, counts after merging and remained to surely in 96 orifice plates after HAT selections are cultivated 10 days respectively
Surely the hole of the hole count grown/96, is converted into percentage (table 1), wherein cell of the conventional chemical agent revulsion in the 96 hole holes Zhong You27
Remain to stablize growth, and cell of the present invention in the 96 hole holes Zhong You42 remains to stablize growth, the effective integration rate of the two be respectively for
28.1% (27/96), 43.8% (42/96), effective integration rate of the invention are induced higher than conventional chemical agent, significant difference
(＜ 0.05).Illustrate that conventional chemical agent revulsion and the pre-confluent rate of the present invention are although almost the same, but conventional chemical agent induces
The ratio of method merged in vain is higher, and the cell fusion of the present invention by means of SPA because mediating double antibody capture, is a kind of special
Property fusion, so its effective integration rate is just high.
(C) influence that chemical inducer toxicity passes on fused cell：Conventional chemical agent revulsion and of the invention are melted
It closes cell to be seeded in for the first time in 96 orifice plates after HAT selections culture 10 days, when most cell clone growth area reaches 1/10
When cell hole, 5 cell holes are respectively selected, culture supernatant is gone, marks position, the size of cell strain growth, use sterile under microscope
Pipette tips mark position draw cell clone, set it is new have in the culture hole of complete medium, then doubling dilution arrives successively
10th hole next, 37 DEG C, the interior culture of 5%CO2 incubators about 1 week, microscopically observation cell growth status (waits for that cell clone is given birth to
When 1/10 or more hole floor space, the other biological characteristic of also desirable cell or culture supernatant detection fusion cell), count
There are cell growth hole and the total hole count of cell inoculation (50 hole), is converted into percentage, as a result conventional chemical agent revulsion is in 50 holes
There are 15 holes to grow cell, the present invention grows cell in the 50 hole holes Zhong You25, and it is respectively 30% and 50% to calculate percentage；By upper method
And then repetition passage experiment is done, the result of wherein 2nd generation is respectively 60% and 76%；The result in the 3rd generation is respectively 80% He
86%, from test result (table 2) it is found that conventional chemical agent revulsion and the present invention are in the passage of 1st generation and 2nd generation, the two
Fused cell pass on the significant difference of growth rate, in the passage in the 3rd generation, the fused cell of the two passage growth rate without
Significant difference, illustrate the cell fusion of the present invention because mediating double antibody capture myeloma cell and B cell by means of SPA thus
The two is set to merge, and contained cytotoxic chemical derivant (PEG) is reduced to 25%, it is very low to cytotoxicity, so passing on
Die during initial cell it is less.And in the cell fusion of conventional chemical derivant revulsion, contained cytotoxic chemical induction
A concentration of the 50% of agent (PEG), larger to cytotoxic effect, so while cell merges, but fused cell is because by poison
Property effect and be easy to passage die during initial.
Compared with 1 conventional chemical agent revulsion of table mediates the cell confluency of dual anti-prize law with SPA
2 conventional chemical agent revulsion of table mediates the influence that dual anti-prize law PEG concentration passes on fused cell with SPA
Claims (3)
1. a kind of SPA mediates the hybridoma technology of double antibody capture, which is characterized in that thin with anti-myeloma cell antibody and anti-B
Born of the same parents' antibody is separately connected myeloma cell and B cell, with SPA connection anti-myeloma cell antibody and anti-B cell antibody, specifically
Property, proportionally furthering is fused the distance of cell, and then under the action of IFN-γ, concanavalin A and low concentration PEG, makes
After birth contacts and fusion, improves effective integration rate and passage success rate.
2. a kind of SPA according to claim 1 mediates the hybridoma technology of double antibody capture, which is characterized in that pre-confluent
Agent includes (1) basal liquid：A concentration of 4.6 μ g/L IFN-γ, 2.5 μ g/ml concanavalin As are incorporated in DMEM culture mediums；(2)
Solution A：The anti-B cell antibody of the potency such as supplying and B cell in basal liquid；(3) second liquid：Supplying and myeloma in basal liquid
The anti-myeloma cell antibody of the potency such as cell；(4) third liquid：Supplying and B cell or myeloma cell or corresponding in basal liquid
The SPA of the potency such as antibody.
3. a kind of SPA according to claim 1 mediates the hybridoma technology of double antibody capture, which is characterized in that pre-confluent
Processing method is to be separately added into B cell and myeloma cell in first centrifuge tube and second centrifuge tube with 10: 1-5: 1 ratio,
1000r/min centrifuges 5min, discards supernatant, and gently beats tube bottom to cell grainless and precipitates, then adds in first centrifuge tube
2ml solution As, in second centrifuge tube plus 2ml second liquid, 37 DEG C 1 hour, by the suspension mixing of first, second centrifuge tube, 37 DEG C 1 hour, then
Be added the third liquid, 37 DEG C 1 hour, 1000r/min centrifuges 5min, discards supernatant, and gently beats tube bottom to cell grainless and precipitates.
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