CN109627328A - Method for preparing monoclonal antibody based on Raman spectrum and microlayer model technology - Google Patents
Method for preparing monoclonal antibody based on Raman spectrum and microlayer model technology Download PDFInfo
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- CN109627328A CN109627328A CN201811650388.4A CN201811650388A CN109627328A CN 109627328 A CN109627328 A CN 109627328A CN 201811650388 A CN201811650388 A CN 201811650388A CN 109627328 A CN109627328 A CN 109627328A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
Abstract
The invention discloses the method for preparing monoclonal antibody based on Raman spectrum and microlayer model technology, including building microfluidic platform and corresponding detection probe, high throughput detects the antibody content in microlayer model containing cell, and the hybridoma to be formed is merged with myeloma containing immunocyte after single antigen-immunized animal or immunocyte in microlayer model.The present invention provides the novel technical method of ultra high efficiency for the preparation of monoclonal antibody, and the monoclonal screening period is greatly shortened, at geometric multiple improves antibody screening ability.
Description
Technical field
The present invention relates to a kind of cell sorting for monoclonal antibody preparation and the technical methods of identification.
Background technique
Monoclonal antibody technique is the important tool of modern life science and medical research and treatment, Koehler in 1975
External hybridoma technology has been founded with Milstein, has obtained the monoclonal antibody in mouse source, the technology was in acquisition promise in 1984
Bell's Medicine and physiology prize.1986, U.S. FDA had approved first monoclonal antibody drug listing, away from the present fast 30
Year.It goes through to list with antibody drug currently, the whole world shares the treatment more than 40, achieves over tens billion of dollars of pin every year
Sell volume.Antibody drug exploitation upsurge is formd in the international and country.
Huge market prospects and existing technical problem and barrier and deposit reality inevitably cause antibody drug
New round technological revolution, and its result will change the market structure of antibody drug without doubt.Antibody is either as inspection
Test agent, or as the therapeutic agent with great market prospect.Firstly, we require to find monoclonal by screening it is anti-
Body.Traditional method for preparing monoclonal antibody is mainly hybridoma technology.This is current comparative maturity, and technical difficulty is relatively low,
Most of laboratories are still in the method used.This method is still that preparation monoclonal antibody is most important in field of scientific study
Method, and in terms of the research and development of therapeutic monoclonal antibodies, although having largely based on humanized antibodies such as display technique of bacteriophage
Technology, but largely listed by the therapeutic antibodies that this method is researched and developed due to existing, consider further that its stability and broad incorporation,
It is still the mainstream technology of this field by hybridomas producing monoclonal antibody technique.
Certain genetic engineering recombinant expression antibody technique is highly developed, as can anti-to get arriving not by hybridoma technology
The gene order of body is more convenient for drug development process.
Basic principle using hybridoma technology preparation monoclonal antibody is according to following three principles: a kind of 1. lymph
Cell clone only generates a kind of antibody;2. the hybridoma that cell-fusion techniques generate can keep both sides' parental cell
Characteristic;3. remedying mechanism using metabolic deficiency filters out hybridoma, and carries out cloning, then mass propgation is proliferated, system
Standby required monoclonal antibody.
By having screening process twice, being using selection for the first time in hybridomas producing monoclonal antibody preparation process
Property culture medium selects hybridoma；Second is exactly further to select the hybridoma that can generate the antigen-specific antibodies needed
Cell.Programmed screening process is extremely complex, needs a large amount of manpower to complete the mask work of subcellular clone.
The following are the summaries to screening technique principle twice:
The principle and method screened for the first time:
After cell fusion, the selectivity culture of hybridoma is the key that screen for the first time.The HAT generallyd use is selectively trained
Nutrient solution is that hypoxanthine, aminopterin and thymidylic acid are added in common animal cell culture liquid.It is cultivated in HAT
The D approach of the fusion product of the B cell not merged in liquid and two B cells is blocked by aminopterin, though S approach is normal, because
Lack the proliferative capacity in culture solution in vitro, it generally can be dead at 10 days or so.For myeloma cell and itself fusion
For cell, since the myeloma cell generallyd use is that hypoxanthine-guanine monophosphate nucleotidyltransferase (HGPRT) defect is thin
Born of the same parents, therefore itself do not have S approach, and D approach is blocked by aminopterin again, thus can not be proliferated in HAT culture solution and very
It is fast dead.The only hybridoma that mutually merges with B cell of myeloma cell not only with the S approach of B cell, but also has bone
The characteristic of myeloma cells culture solution long-term proliferation in vitro, therefore can selectively be survived in HAT culture solution, and constantly
Proliferation.
The principle and method of programmed screening:
In the production process of monoclonal antibody, since the specificity of bone-marrow-derived lymphocyte is different, for the first time through HAT culture solution
There are diversity for the antibody that the hybridoma filtered out generates, it is necessary to carry out programmed screening, ability to hybridoma group
Select the hybridoma that there is specificity for target antigen.Postsearch screening generallys use limiting dilution assay, and hybridoma is thin
More times of born of the same parents dilutions, are seeded on porous cell culture plate, and every hole is made to be no more than a cell, allow it to rise in value by culture.Then
The specificity (common ELISA method) for the antibody that cell is secreted in each hole supernatant is detected, supernatant can be in conjunction with specific antigen
Culture hole be positive hole.Cell can't be ensured of from individual cells in positive hole, need to continue limiting dilution, and one
As repeat 3-4 time, until firmly believing in each hole that value-added cell is monoclonal cell.Programmed screening is also to identify specific parent
With the process of power.
Limiting dilution assay is most common method for screening hybridoma, is all being used in many laboratories.But
It is, as relatively old technology, to be no longer satisfied current demand.Method itself has the shortcomings that many:
1, the step of manual operation, operation and repetition, is too many, inevitable to be easy error.2, speed is slow, low efficiency.It can only protect
There is cell growth in the hole of card 30-40%.For high flux screening, it cannot meet the requirements.3, it cannot be guaranteed that monoclonal, needs
Subclone 3-4 times, so, it is artificial and time-consuming all more.4, limiting dilution is before ELISA detection, in this case it is not apparent which clone
Meet the requirement of antigentic specificity.It is therefore desirable to will at least do an ELISA identification to all clones.
In addition to limited dilution method, now relatively advanced cell sorting techniques are also widely used.It is exactly generally thin
Born of the same parents' sorter or drain cell technology screen hybridoma.Its principle is that first cell scatter, without cell
Conglomeration.By in cell surface or cell inner mark fluorescence signal.When individual cells individually pass through glass fiber under pressure
When tubule, instrument can detect the fluorescence signal intensity of individual cells automatically.Fluorescence signal intensity reacts this cell
Corresponding antibodies expression.Finally, the cell of high fluorescence signal intensity is collected, then obtain high level expression by limiting dilution
Monoclonal;Or the single hybridoma that unicellular collector collects high level expression also can be used.
This technology also uses in many laboratories, but screens principle according to it, there is some inevitable defects.
1, this screening criteria to cell is not also to detect antibody expression amount for real individual cells.
2, due to having to cell surface or inside using fluorescent marker, so, generally it is mainly used for intracellular expression
Albumen or film expression albumen.For the hybridoma of secreting, expressing, using with limitation.
3, what is specifically detected is the fluorescence intensity of individual cells at some time point.So testing result is easy by cell
The influence in period.Moreover, the expression of individual cells can not react the expression water of post-production phase cell colony very well
It is flat.
4, streaming screening technique is that all cells strong to fluorescence signal make division, and the later period needs the cell number of secondary screening
Mesh is still very much, and downstream workload is still bigger.
5, due to using pressure operation to individual cells, shearing force can reduce cell survival rate.So low survival rate also at
For a major defect of this technology.
5, streaming screening needs just to can ensure that cell will not pollute to the abundant cleaning and sterilizing of pipe-line system, the risk of pollution compared with
It is high.
It is similar to above-mentioned technical method, and occur using the cellifugal method of semisolid culturemedium point, then pass through machinery
Arm and u-turn sort mode to the positive cell clone that specific phosphor dot just operates.But this method disadvantage in use
Clearly, first hybridoma want can on semisolid culturemedium well-grown, and many cells are to be unable to reach this requirement
's.Secondly, the size and density of clone have a significant impact for the accuracy for automating picked clones, clone is too small too close, all
It will affect identification and picking, and then reduce the monoclonicity for choosing clone.
With technological progress, also there is an urgent need to a kind of more advanced screening techniques for we.It can satisfy high throughput, automatically
Change, efficient requirement.
It is growing in view of the widely applied needs to monoclonal antibody, however the operation of conventional hybridization tumor method has very
Complexity, especially single plantization select the process grown, and not only need to take a significant amount of time, while must also consume many manpowers, and grasp
The proficiency of author also will affect as a result, therefore often the success or failure of experiment are decided by this step, even by fluorescent technique knot
Mechanical arm picking is closed, is not still the technology for directly detecting individual cells expression amount of antibody, technical complexity is excessively high, and timeliness
Property is but very low.
Summary of the invention
It is an object of that present invention to provide one kind can directly detect individual cells antibody expression amount, and as measurement
Standard sorts fused cell, obtains the single cell clone that expression specific antigen corresponds to antibody.
The major technique used in the present invention is the Surface enhanced Raman spectroscopy (Surface- based on single cell analysis
Enhanced Raman spectroscopy, SERS) and Microfluidic droplet technology (Microfluidic Droplet), including
Following steps:
S1: preparing specific antigen and usually share three times for animal, the immune of animal to be immunized, take spleen after last time is immune,
Prepare splenocyte suspension；
S2: cell fusion is carried out using the splenocyte in myeloma cell and step S1 and prepares hybridoma；
S3: microfluidic platform is built
S4: the probe with Raman signal is prepared；
S5: Raman spectrum detection；
S6: unicellular culture and sequencing；
Based on above description, wherein step S1 and S2 is the conventional method of monoclonal antibody preparation.Hybridoma fusion skill can not also be passed through
Art makes cell immortality, directly by the splenocyte suspension in step S1, for executing S3 step.The cell that final S6 step obtains
It is sequenced to obtain antibody sequence.
Step S3 described above is comprised the steps of:
S3A: negative photoresist being spun on clean silicon wafer, and carries out patterned exposure to 385 nm LED arrays
Light source.Therefore, the microchannel structure with mask pattern is transferred to film.Then, by Sylga dimethyl silicone polymer and crosslinking
Agent (ratio 10:1) is mixed and is thoroughly deaerated, and is poured upon on photoetching agent pattern, and solidify at 80 DEG C at least 3 hours.
PDMS duplicate is removed from chip.Before PDMS is adhered on glass slide, PDMS and the oxygen of glass surface etc. are carried out
Gas ions activation；
S3B: it is bonded under high pressure and heating condition with upper-part.Then with Aquapel solution filling microfluidic channel to improve
Their wetabilitys to fluorinated oil.Internal diameter is 0.8mm, and outer diameter is that the polyethylene pipe of 1.6mm is connected to channel by syringe pump；
Step S4 described above is comprised the steps of:
S4A: can specifically bind antibody Fc fragment staphylococcal protein A (SPA) coupled bead (MNs@SPA, about 500nm),
It is modified with Raman signal report molecule on it；
S4B: the antigen of immune animal is coupled nano grain of silver (AgNPs@Antigen, about 48nm).
Step S5 described above is comprised the steps of:
S5A: high-throughput two drops of grease are realized using micro-fluidic chip, contain an independent cell in each oil droplet
With the probe (as shown in Figure 2) synthesized in two kinds of S4 steps, then measured by the capture of collection room array channel for SERS.
S5B: in the presence of the antibody of single clone cell secretion, AgNPs@Antigen can be by above
Antigen recognizing antibody and antibody are in conjunction with the surface SPA of MNs@SPA, structure as shown in Figure 2.
S5C: it is close due to AgNPs, so that the SERS signal of the Raman reporter molecules on MNs is significantly increased
By force.In addition, SERS signal generation is further amplified from the design of induced by magnetic field building-up effect, SERS signal can further improve
(Fig. 4).In addition, for the package of the secreted monoclonal antibody expressed of cell, so that increase of the analyte with the reaction time
And build-up effect.Due to dual signal enhancing and summation, a small amount of antibody of individual cells secretion can be detected, and detection limit can
Reach 1fg.
In step S6 described above, the drop separations of default testing requirements will be reached with Raman signal to cell
It is to be checked in culture tool.
The present invention has the advantages that
1. the time needed for substantially shortening monoclonal antibody preparation, the stage the most time-consuming is exactly to express antigen phase in monoclonal antibody preparation
Answer the separation phase of antibody monoclonal cell, it usually needs 2-3 months, and more than ten hour is only needed in the present invention；
2. the gene order as only needed antibody, then can save hybridoma cell fusion and monoclonal separation process；
3. can be realized high throughput, can will isolated all cell clones for generating antibody, provided more for drug development
More options；
Human cost and antibody price will be greatly reduced in this method, bring industry transformation.
Detailed description of the invention
Two kinds of immune particle schematic diagrames of Fig. 1.Antigen is coupled nano grain of silver (AgNPs@Antigen, about 48nm) and SPA is even
Join magnetic bead (MNs@SPA, about 500nm).The modification of magnetic bead surfaces Raman reporter molecules is indicated with star-like.
Immune identification action between Fig. 2 antibody/antigen is connected to the surface action schematic diagram of SPA coupled bead.
Fig. 3 prepares platform schematic diagram based on the monoclonal antibody of Raman spectrum and microlayer model technology.
It is Raman detection signal graph on the left of Fig. 4, right side is antibody content and Raman reporter molecules corresponding relationship.
Specific embodiment
Surface enhanced Raman spectroscopy and Microfluidic droplet technical appraisement individual cells antibody expression based on single cell analysis
It measures and as the method that measurement standard sorts fused cell, in following implementation content, with human epidermal growth factor
Sub- receptor 2(HER2) extracellular domain be antigen, prepare the monoclonal antibody of anti-HER2.The following steps are included:
S1: preparing specific antigen and usually share three times for animal, the immune of animal to be immunized, take spleen after last time is immune,
Prepare splenocyte suspension；
Using the HER2 extracellular domain of genetic engineering recombinant expression as antigen, the Balb/c mouse of 6 week old is immunized, three times addition assistant
Agent is immune, and immunization time is separated by three weeks, and third day takes spleen after last time is immune, tests for cell fusion；
S2: cell fusion is carried out using the splenocyte in myeloma cell and step S1 and prepares hybridoma；
Myeloma cell is prepared, RPMI1640 culture medium culture mouse myeloma, serum-concentration 15%, when cell is grown to are utilized
It when logarithmic growth phase posterior segment, is passed on according to the ratio of 1:4, is handled during passage using 8-anaguanine, keep living
Sensibility of the cell to HAT.Use Peritoneal Cells of Mice as feeder cells simultaneously.
The following are the methods that present embodiment carries out cell fusion: by myeloma cell and splenocyte according to the ratio of 1:10
It is mixed, as 50mL centrifuge tube, is rinsed using serum-free nutrient deficiency culture medium, is centrifuged 800g ten minutes, abandoned
Supernatant, cleans residual liquid, and slight vibration loosens cell precipitation；40 DEG C of 50%PEG is heated in 45 seconds with suction pipe
(pH8.0) in liquid, the defect culture medium for being preheated 30mL in two minutes with suction pipe terminates the effect of PEG, stands at 25 DEG C
10 minutes；1000g is centrifuged 5 minutes, discards supernatant, and 5mLHAT culture medium is added, gently blows and beats sedimentation cell, makes these cells
It suspends and mixes, the HAT culture medium containing abdominal cavity cell is then added to 100mL addition feeder cells；Then by culture plate
37 DEG C are placed in, 6%CO2In incubator.The HAT culture medium that half was displaced in the 6th day is replaced after eight days with HT culture medium
HAT culture medium still has hundreds of cell survivals, is ready for the detection of Microfluidic droplet Raman spectrum and sorting.
S3: microfluidic platform is built
S4: the probe with Raman signal is prepared；
S5: Raman spectrum detection；
The following are Fig. 1 of micro-fluidic detection platform and Raman spectrum testing principle
It is prepared for two kinds of immune particles (Fig. 1) first, antigen is coupled nano grain of silver (AgNPs@Antigen, about 48nm) and SPA is even
Join magnetic bead (MNs@SPA, about 500nm).Magnetic bead surfaces also modify (star-like expression) with Raman reporter molecules.
High-throughput two drops of grease are realized using micro-fluidic chip, contain an independent cell in each oil droplet
With two kinds of immune particles, then measured by the capture of collection room array channel for SERS.The cell secreting, expressing antibody the case where
Under, AgNPs@Antigen can be connected to surface (such as Fig. 2 of MNs@SPA by the immune identification action between antibody/antigen
It is shown).It is close due to AgNPs, so that the SERS signal of the Raman reporter molecules on MNs is significantly enhanced.In addition,
SERS signal generation is further amplified from the design of induced by magnetic field building-up effect, can further improve 75 times of SERS signal (Fig. 3).
In addition, package of the antibody molecule constantly expressed in separation drop is so that analyte accumulates effect with the increase in reaction time
It answers.Due to dual signal enhancing and summation, a small amount of antibody of individual cells secretion can be detected.
First verify that the method for sensing.The MNs@SPA+ AgNPs@Antigen of various concentration will be wrapped up in drop, carry out
Detection.As a result as shown in figure 4, antibody content is linearly related to Raman reporter molecules, it was demonstrated that this method is feasible.In drop,
Two various particles are wrapped up, and drop is added in antibody, concentration changes from low to high, and SERS testing result shows Raman reporter molecules
1178 cm-1 of characteristic peak of AAD.Regression analysis, R are carried out to detectable substance concentration to peak intensity2=0.993, it is preferable linear junction
Fruit.In next implementation process, we are had detected in the drop of a large amount of individual cells, determine that detectable concentration can reach
1.0 the limit of fg/mL.
S6: unicellular culture and sequencing, the splenocyte that can get overexpression protection body in the step (do not carry out the behaviour of S2 step
Make) or hybridoma, it can be cultivated, obtain lot of antibodies, or carry out unicellular sequencing, obtain antibody sequence.
Specific embodiment is that invention is further explained, but the invention is not limited to these specific embodiment party
Formula.
Claims (3)
1. the method for preparing monoclonal antibody based on Raman spectrum and microlayer model technology, which is characterized in that include the following steps;
S1: preparing specific antigen and usually share three times for animal, the immune of animal to be immunized, take spleen after last time is immune,
Prepare splenocyte suspension；
S2: cell fusion is carried out using the splenocyte in myeloma cell and step S1 and prepares hybridoma；
Wherein step S1 and S2 is the conventional method of monoclonal antibody preparation；
It can not also make cell immortality by hybridoma fusion techniques, directly by the splenocyte suspension in step S1, for holding
Row S3 step, the cell that final S6 step obtains are sequenced to obtain antibody sequence；
S3: microfluidic platform is built
S3A: negative photoresist being spun on clean silicon wafer, and carries out patterned exposure to 385 nm LED arrays
Light source;Therefore, the microchannel structure with mask pattern is transferred to film;Then, by Sylga dimethyl silicone polymer and crosslinking
Agent (ratio 10:1) is mixed and is thoroughly deaerated, and is poured upon on photoetching agent pattern, and solidify at 80 DEG C at least 3 hours；
PDMS duplicate is removed from chip；Before PDMS is adhered on glass slide, PDMS and glass surface are carried out
Oxygen plasma activation；
S3B: being bonded under high pressure and heating condition with upper-part, then with Aquapel solution filling microfluidic channel to improve
Their wetabilitys to fluorinated oil；Internal diameter is 0.8mm, and outer diameter is that the polyethylene pipe of 1.6mm is connected to channel by syringe pump；
S4: the probe with Raman signal is prepared；
S4A: can specifically bind antibody Fc fragment staphylococcal protein A (SPA) coupled bead (MNs@SPA, about 500nm),
It is modified with Raman signal report molecule on it；
S4B: the antigen of immune animal is coupled nano grain of silver (AgNPs@Antigen, about 48nm);
S5: Raman spectrum detection；
S5A: high-throughput two drops of grease are realized using micro-fluidic chip, contain an independent cell in each oil droplet
With the probe (as shown in Figure 2) synthesized in two kinds of S4 steps, then measured by the capture of collection room array channel for SERS；
S5B: in the presence of the antibody of single clone cell secretion, AgNPs@Antigen can pass through antigen above
Identify antibody and antibody in conjunction with the surface SPA of MNs@SPA, structure as shown in Figure 2；
S5C: it is close due to AgNPs, so that the SERS signal of the Raman reporter molecules on MNs is significantly enhanced；
In addition, SERS signal generation is further amplified from the design of induced by magnetic field building-up effect, SERS signal can further improve
(Fig. 4)；In addition, for the package of the secreted monoclonal antibody expressed of cell, so that increase of the analyte with the reaction time
And build-up effect；Due to dual signal enhancing and summation, a small amount of antibody of individual cells secretion can be detected, and detection limit can
Reach 1fg；
S6: unicellular culture and sequencing will have Raman signal to reach default testing requirements in step S6 described above
Drop separation into cell culture tool, it is to be checked.
2. the method for preparing monoclonal antibody according to claim 1 based on Raman spectrum and microlayer model technology, wherein
Step S4 prepares the probe with Raman signal, other materials equally can be used prepares material as probe.
3. the method for preparing monoclonal antibody according to claim 1 based on Raman spectrum and microlayer model technology, wherein
Step S4 prepares the probe with Raman signal, other substances that can be interacted with detection target protein can be used.
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| CN111948386A (en) * | 2020-09-03 | 2020-11-17 | 吉林大学 | Raman homogeneous detection method for recognizing and combining antigen by using antibody |
| CN113189078A (en) * | 2021-03-04 | 2021-07-30 | 吉林大学 | High-throughput screening method for targeted drugs |
| WO2023133895A1 (en) * | 2022-01-17 | 2023-07-20 | 深圳华大生命科学研究院 | Method for obtaining information on nanoantibody specifically recognizing antigen |
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Application publication date: 20190416 |