CN108250287A - A kind of easy method for finding cell-specific antigens - Google Patents
A kind of easy method for finding cell-specific antigens Download PDFInfo
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- CN108250287A CN108250287A CN201810081245.XA CN201810081245A CN108250287A CN 108250287 A CN108250287 A CN 108250287A CN 201810081245 A CN201810081245 A CN 201810081245A CN 108250287 A CN108250287 A CN 108250287A
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Abstract
This application discloses a kind of easy methods for finding cell-specific antigens.Include the following steps：A. anti-normal cell or the serum of normal structure are obtained；B. animal is handled with the step A antiserums obtained or the polyclonal antibody being purified；C. the processed animals of step B are immunized with corresponding cancer cell or cancerous issue；D. the B cell of animal after separating step C is handled；E. the B cell after detaching is merged with myeloma cell, obtains cell strain of monoclonal antibody；Monoclonal antibody is prepared with cell strain of monoclonal antibody；F. the cell pyrolysis liquid for the monoclonal antibody affinity purification cancer cell that step E is obtained, obtains antigen protein in connection.Using new method of the present invention, do not have to or do not have to subsequent negative screening process substantially.This undoubtedly greatly increases the efficiency of screening.
Description
Technical field
The present invention relates to molecular biology antigen-antibody screening technique field, more particularly to a kind of easy searching cell-specific
The method of property antigen.
Background technology
In the biological therapy scheme of current tumour, immunization therapy is the hot spot always studied and applied.Generally
For, there are mainly two types of main modes at present.A kind of is based on cellular immunity, that is, killer T cell is allowed to identify and is killed
Cancer cell；Another is based on humoral immunity, that is, obtains the specific antibody of tumour, then passes through antibody activating complement way
Diameter or the killing cancer cell of the drug specificity by being coupled on antibody.But whether which kind of immunotherapy, allows immune system to know
Other cancer cell is a step of most critical.
But cancer cell is a kind of neoplasm cell for immunity of organism monitoring of having escaped, it " pretends " as far as possible into normal
Cell is identified and is removed to avoid by body immune system, therefore is wanted immune system identification cancer cell and become extremely difficult.
For tumour antibody treatment, the step of most critical is the monoclonal antibody for obtaining cancer cell specificity.At present
Tumor specific antibody is obtained there are mainly two types of mode：
The first, is directly immunized mouse, and obtain the monoclonal antibody for resisting the cancer cell with certain cancer cell or its extract, so logical
It crosses and again these antibody is carried out with negative screening with control cell (such as corresponding normal cell), finally obtain specific antibody；
Second, the albumen of cancer cell specificity, especially memebrane protein are obtained by molecular biology method first, then
It goes immune with this albumen and obtains monoclonal antibody.
However both methods has many problems.Cancer cell is the cell that immune system monitors of having escaped as previously described,
So while from molecular scale, cancer cell has many differences with normal cell really, but this difference is not sufficient to
Activate the immune system of body.So if obtaining antibody with first method, the present invention is generally required from thousands of or even tens of thousands of
The antibody of a cancer cell specificity can be screened in a clone.Also because of this defect, this method quilt substantially
It eliminates.Second method is also the method for current mainstream, and people have obtained some antibody and answered by this method
With.
But present invention understands that, not a kind of cancer cell of tumour is bound to express this antigen, even if in a tumour
Nor all cancer cells can all express certain specific antigen in tissue.So this antibody can only be identified and be killed
Wound expresses the cancer cell of this antigen, therefore the cancer cell without expressing this antigen but is screened out simultaneously neoplasm.Cancer
This high variability of cell is also one of the reason of cancer is difficult to treat.
Just because of variability and the diversity of cancer cell, more and more experts think, for immunotherapy,
Only several or more antibody combined treatments, i.e. " cocktail therapy " are possible to solve the problems, such as this.But as previously mentioned,
Want that it is extremely difficult to obtain how species specific antibody by the both methods of current mainstream.Therefore, it is how as high as possible
Effect ground obtains the antibody of tumour-specific, has become the bottleneck problem of tumour antibody treatment.
Invention content
The technical problems to be solved by the invention are, provide a kind of easy method for finding cell-specific antigens.
The method of the present invention obtains the monoclonal antibody of 11 kinds of anti-HepG2.These monoclonal antibodies have HepG2 the identification of specificity, and fail to see
Other or basic nonrecognition HH cell.Meanwhile tissue-derived cancer cell compares with other, these monoclonal antibodies all have spy to HepG2
The identification of the opposite sex.
In order to solve the above technical problems, the present invention provides a kind of easy method for finding cell-specific antigens, including
Following steps：
A. anti-normal cell or the serum of normal structure are obtained；
B. animal is handled with the step A antiserums obtained or the polyclonal antibody being purified；
C. the processed animals of step B are immunized with corresponding cancer cell or cancerous issue；
D. the B cell of animal after separating step C is handled；
E. the B cell after detaching is merged with myeloma cell, obtains cell strain of monoclonal antibody；Use Monoclonal Antibody Cell
Strain prepares monoclonal antibody；
F. the cell pyrolysis liquid for the monoclonal antibody affinity purification cancer cell that step E is obtained, obtains antigen in connection
Albumen.
The cancer cell is preferably liver cancer cells.
The method for obtaining unknown specific antigen cancer cell specific antibody, preferably further includes：
G. the antigen protein that mass spectrometry procedure F is obtained.
The step A may further include：
A1. cell is collected；
A2. gravity treatment cell；
A3. serum is obtained after being immunized.
The sub-step A1 may further include：
When normal cell culture is expanded to density and reaches 75~85%, culture solution is sucked；First remaining culture is washed away with PBS
Then base handles cell with PBS/8-12mM EDTA again, make cell detachment and finally piping and druming collection cell.
The sub-step A2 may further include：
800~1200 turns, after centrifugation in 3-5 minutes, cell precipitation is dispelled with 1% paraformaldehyde, and room temperature fix 0.3~
0.7 hour, paraformaldehyde is then washed away by centrifugation, and with PBS gravity treatment cells, after cell count, be finally stored in -20 DEG C.
The sub-step A3 may further include：
1,000,000 cell constant volumes is taken to shake in 200 μ l PBS and with 200 μ l complete Freund's adjuvants or incomplete Freund's adjuvant vortex
Swing mixing；Immune 5-7 week old mouse, 1,000,000 cellular immunities of every mouse, immunization interval 2 weeks are 2-4 times common；Last time is exempted from
Epidemic disease is taken a blood sample after 5-15 days collects serum.
The step C preferably further includes：
C1. cell is collected；
C2. gravity treatment cell；
C3. B cell is obtained after being immunized.
The sub-step B1 preferably further includes：When cancer cell culture is expanded to density and reaches 78-82% or so, suck
Culture solution；Remaining culture medium first is washed away with PBS, then handles cell with PBS/7-13mM EDTA again, makes cell detachment and most
Cell is collected in piping and druming eventually.
The sub-step B2 preferably further includes：
900~1100 turns, after centrifugation in 3-5 minutes, cell precipitation is dispelled, and room temperature is fixed with 0.5-1.5% paraformaldehydes
0.3~0.7 hour, paraformaldehyde is then washed away by centrifugation, and with PBS gravity treatment cells, after cell count, be finally stored in-
20℃。
Advantageous effect of the present invention includes：
1) 11 kinds of antibody that the present invention filters out directly are screened with HepG2 cells, that is to say, that not by HH
The negative screening of cell or other cells.Using new method of the present invention, do not have to or do not have to subsequent negative screening process substantially.This nothing
Doubt the efficiency for greatly increasing screening.
2) the method for the present invention is particularly suitable for obtaining unknown specific antigen cancer cell without being immunized with known antigens
Specific antibody.
3) present approach provides a kind of simple method for finding cell-specific antigens, this 11 kinds of Dan Ke can be used
Grand antibody removes the cell pyrolysis liquid of affinity purification HepG2, and the present invention so has just obtained the albumen that can be combined with them.Then
It is gone to analyze these albumen with mass spectrum, it is possible to which it is what to know these albumen, facilitates further further investigation.
Description of the drawings
Fig. 1 is removes cancer cell and corresponding normal cell common antigen described in the embodiment of the present invention and to obtain anticancer thin in vivo
The schematic diagram of born of the same parents' monoclonal antibody；
Recognition capability comparison diagram of the serum to HH cell and HepG2 that Fig. 2 is anti-HH cell described in the embodiment of the present invention；
Fig. 3 is 11 kinds of monoclonal antibody specificity recognition capability column diagrams described in the embodiment of the present invention；
Fig. 4 compares HepG2 specific recognitions with monoclonal antibody of the present invention for other types of cell described in the embodiment of the present invention
Figure；
Fig. 5 is removes cancer cell and corresponding normal cell common antigen and is finally resisted in vitro described in the embodiment of the present invention
The schematic diagram of cancer cell monoclonal antibody；
Fig. 6 is that the monoclonal antibody specificity that in vitro method described in the embodiment of the present invention obtains identifies HepG2 ability column diagrams.
Specific embodiment
The present invention is described in detail with reference to embodiment.To make the objectives, technical solutions, and advantages of the present invention clearer, bright
Really, developing simultaneously referring to the drawings, the present invention is described in more detail for embodiment, but the invention is not limited in these embodiments.
Unless otherwise instructed, the raw material in the embodiment of the present invention and catalyst are bought by commercial sources.
In the immunotherapy processes of numerous tumours, the monoclonal antibody for obtaining specific anti-cancer cell is always therein
One key factor.But regrettably up to the present, there are no it is a kind of can be in the method for the high frequency zone antibody.The present invention
Provide a kind of efficient method for obtaining inhibiting tumor cell monoclonal antibody.The core design thought of the method for the present invention is, with anti-phase
Corresponding normal cell antibody directly removes the antigen rather than carry out in vitro big that cancer cell and normal cell share in vivo
This method is known as " antibody filter " by the screening of scale, the present invention.By this method, the present invention is with hepatocellular carcinoma H22
For model embodiment, the monoclonal cell strain of 11 anti-HepG2 is obtained.In the experiment of the present invention, present invention discover that these
Antibody has HepG2 specific recognition, and normal liver cell (HH cell) and other tissue-derived cells are failed to see substantially
Not.Pass through the experiment of the present invention, it was demonstrated that this be it is a kind of can be in the method for high frequency zone inhibiting tumor cell monoclonal antibody, and future
This method is combined the treatment that would be possible to applied to cancer with humanization technologies or drug coupling technology.
As shown in Figure 1, for cancer cell and corresponding normal cell common antigen are removed described in the embodiment of the present invention in vivo and is obtained
Obtain the schematic diagram of inhibiting tumor cell monoclonal antibody.
Wherein step is：1, mouse is immunized with normal cell；2, acquire anti-normal cell serum；3：Antiserum is injected into
Other mouse；4, meanwhile, with the corresponding carcinoma cell immunization mouse；5, collect the splenocyte of the mouse；6, cell fusion；7,
Screening resists the cell strain of monoclonal antibody of the cancer cell.
The new method of the present invention obtains the monoclonal antibody of 11 kinds of anti-HepG2.These monoclonal antibodies have HepG2 specificity
Identification, and nonrecognition or basic nonrecognition HH cell.Meanwhile tissue-derived cancer cell compares with other, these monoclonal antibodies are all
There is the identification of specificity to HepG2.
First, the full cell of normal cell (such as HH cell) of the invention or full cell pyrolysis liquid or extracting
The memebrane protein gone out just obtains the serum of anti-HH cell as mice immunized with antigen, the in this way present invention.The subsequent present invention will be this
Antiserum or the new mouse not being immunized of the polyclonal antibody injection being purified, at the same with corresponding cancer cell (such as
HepG2 it) goes that the mouse is immunized.Because having there is the antibody of anti-HH cell in the Mice Body at this time, in HepG2 cells
The antigen shared with HH cell can be identified by anti-HH cell antibody, and finally be swallowed by macrophage or other phagocytes.
HepG2 antigens remaining in this way are exactly the specific antigen of the cell.These antigens can activate the immunity system of the mouse,
And cause hyperplasia and the differentiation of corresponding bone-marrow-derived lymphocyte.Finally, (bone-marrow-derived lymphocyte accounts for very big the splenocyte of the present invention separation mouse
Ratio) and with SP2/0 cell fusions, finally obtain cell strain of monoclonal antibody.As mentioned previously, because the anti-HH cell of injection
Serum has removed the antigen that HepG2 and HH cell are shared, therefore this incomplete antigen can not cause the immune response of body
Also just break up without the increment of corresponding bone-marrow-derived lymphocyte, therefore the cell strain of monoclonal antibody that the present invention finally obtains, only resist
HepG2 specific antigens.This serum or antibody by anti-normal cell of present invention pipe is removed corresponding normal thin in vivo
Born of the same parents share antigen with cancer cell, and the method for finally obtaining cancer cell specific antibody, are called " antibody filter ".
As shown in Fig. 2, the serum for HH cell anti-described in the embodiment of the present invention is to the recognition capability of HH cell and HepG2
Comparison diagram.The figure is the results show that anti-HH cell serum has identical recognition capability to HH cell and HepG2.
The present invention is immunized BALB/c mouse with the fixed HH cell of paraformaldehyde and is finally obtained the anti-of anti-HH cell
Then serum dilutes the serum with 1000 times of PBS/1%BSA, and with the PBS/1%BSA multiples gradient dilution serum, highest
Extension rate is 2,048,000 times.Then the present invention spreads 96 porocyte culture plates with HH cell and HepG2 cell respectively, and examines
The antiserum is surveyed to their recognition capability either with or without difference.The results show that even if extension rate reaches 2,000,000 times, the serum
To the recognition capabilities of both cells, there is no difference.
As shown in figure 3, for 11 kinds of monoclonal antibody specificity recognition capability column diagrams described in the embodiment of the present invention.The figure knot
Fruit shows, the basic nonrecognition HH cell with 11 kinds of monoclonal antibody specificities identification HepG2 that the method for the present invention obtains.
Next, anti-HH cell serum injections (tail vein injection or intraperitoneal injection) are entered 5 new BALB/c by the present invention
Mouse, and with the fixed HepG2 cellular immunities mouse of paraformaldehyde.In immunologic process, the method for the present invention is by periodically noting
Anti- HH cell serum is penetrated to maintain the concentration of its internal anti-HH cell antibody.It is immunized by three-wheel, the method for the present invention takes this small
The spleen of mouse and with SP2/0 cell fusions.The monoclonal cell strain finally obtained with HepG2 cell screenings, and be finally obtained
11 kinds of anti-HepG2 monoclonal antibodies.By detection, this 11 kinds of monoclonal antibody nonrecognition or basic nonrecognition HH cell, still
There is strong identification to HepG2, illustrate the specificity of these monoclonal antibodies.
11 kinds of monoclonal antibodies that the present invention obtains, wherein only HP1, HP2 and HP3 are IgG1 hypotypes, remaining 8 kinds of whole
It is IgM hypotypes.It is interesting that the monoclonal antibody of three kinds of IgG1 hypotypes generally has more HepG2 than remaining monoclonal antibody
Good recognition capability.
As shown in figure 4, HepG2 specificity is known with monoclonal antibody of the present invention for other types of cell described in the embodiment of the present invention
Other comparison diagram.Fig. 4 shows that these monoclonal antibodies have HepG2 more specific identification compared with other kinds of cell.Wherein：1,
HepG2；2,786-O；3, A375；4, DLD-1；5, ScaBER；6, A549；7, H441；8, WI-38.Its ordinate is opposite knowledge
Other ability, and abscissa is above-mentioned various cells.
Other than human liver cell, the present invention also has detected other tissue-derived cancer cells and immortalized cells respectively, including：
786-O, A375, DLD-1, ScaBER, A549, H441 and WI-38.Present invention discover that compared with these cells, this 11 kinds of antibody
There is better recognition capability to HepG2.
Experimental result show that anti-normal liver cell (HH cell) serum is in identification HH cell and liver cancer cells
(HepG2) there is no difference in ability.This result one is can why cancer cell can escape immune system with indirect interpretation
Monitoring, because cancer cell is simulating normal cell as far as possible；In addition can also illustrate why traditional direct exempted from cancer cell
Epidemic disease and the method that obtains monoclonal antibody lacks efficiency, as it means that people need to eliminate a large amount of list with normal cell cross reaction
Clonal antibody cell strain.
In the 11 kinds of monoclonal antibodies obtained in the present invention, 8 are IgM hypotypes.It is generally believed that IgM hypotypes mainly occur
In initial immunity or weak immunologic process.This result on the one hand show HH cell cell surface antigen and HepG2 it is thin
Cellular surface antigen is highly consistent, the antigen that both cells of the anti-HH cell cleaning antibodies that are injected into this way share, and remaining
HepG2 amount of antigen it is too low, it is difficult to effectively activate immunity system.Still further aspect, even in this case, 8 kinds
IgM hypotypes monoclonal antibody has HepG2 more specific identification, and this further demonstrates the reasonability of this method design.It removes
Except this, it has also been found that three kinds of IgG1 hypotypes monoclonal antibodies (HP1, HP2, HP3) are better than greatly the recognition capability of HepG2
The antibody of part IgM hypotypes.This result shows that, the cell surface antigen amount of the corresponding HepG2 of these three antibody be it is enough,
So they induce enough immune responses.Then pass through the Immune Clone Selection of bone-marrow-derived lymphocyte, body is produced to antigen recognizing energy
The stronger IgG1 subclass antibodies of power.
This also prompts the present invention, if this method is further improved, such as advanced optimizes the anti-HH of adjustment
The ratio of the injection volume of cell antibody and immune HepG2 amounts, i.e., be enough to remove what two kinds of cells shared in anti-HH cell antibody
The amount of HepG2 specific antigens is improved under the premise of antigen as far as possible, is possible to obtain so more affine with higher
The antibody of the hypotypes such as IgG1, IgG2a and IgG2b of power.
In addition, the present invention's is full cellular immunity, it is immune that this has meant that many intracellular proteins have also assisted in, this
Permitted to be explained further antibody the why present invention obtains it is less and also it is most of be IgM hypotypes.Therefore in next step, after the present invention
Continuous research association only extracts memebrane protein and is immunized, this can further improve the quality and quantity of specific antibody in theory.
In order to further detect the specificity of these monoclonal antibodies, the present invention has detected hyaline cell with this 11 kinds of monoclonal antibodies
Renal cell carcinoma cell (786-O), melanoma cells (A375), colon cancer cell (DLD-1), transitional cell bladder carcinoma cell line (ScaBER),
Lung carcinoma cell (A549, H441) and people's normal fibroblast (WI-38).The result shows that compared with liver cancer cells (HepG2),
11 kinds of antibody are weaker to their basic nonrecognition or recognition capability.On the one hand this result further illustrates this 11 kinds of monoclonal antibodies
Specificity, on the other hand also illustrate separate sources cell have very big difference on cell surface antigen.This hair simultaneously
The bright cell for noticing HP-5 and can also equally identifying some other sources, perhaps this imply the antigen of HP-5 identifications at other
Some type of cell surface equally has expression.
Present invention obtains 11 kinds of monoclonal antibodies.Although it is IgM hypotypes wherein to have 8, with HH cell and its
His cell, which compares this 11 kinds of antibody, has HepG2 very strong specific binding.Herein it is important to note that this 11 kinds anti-
Body is directly screened with HepG2 cells, that is to say, that not by the negative screening of HH cell or other cells.
In other words, using the new method of the present invention, do not have to or do not have to subsequent negative screening process substantially.This is undoubtedly big
The earth improves the efficiency of screening.Simultaneously as this method with known antigens without being immunized, then it is just particularly suitable for
Obtain the specific antibody of unknown specific antigen cancer cell.
And present approach provides a kind of simple methods for finding cell-specific antigens.It is such as of the invention
The cell pyrolysis liquid of affinity purification HepG2 can be removed with this 11 kinds of monoclonal antibodies, just obtained can be with it by the present invention in this way
The albumen that combines.Then it is gone to analyze these albumen with mass spectrum or other methods, it is possible to which it is what to know these albumen, this nothing
It doubts and facilitates further further investigation.
As previously mentioned, in the Antybody therapy of tumour, monospecific antibody treatment increasingly shows limitation.It is this is because swollen
Knurl has high variability in itself, and the treatment of monospecific antibody has the very big cancer cell that those are not expressed specific antigen may sieve
It elects, and to be likely to be grade malignancy higher for these cancer cells.It is several or more so more and more scientists think
Multiple Antibodies are administered simultaneously, that is, so-called " cocktail therapy ", can reduce the ratio of escape killing cancer cell as far as possible.
But the antibody of tumour-specific how is efficiently obtained exactly where key to the issue.
In the experiment of the present invention, the present invention can screen 11 species specificity monoclonal antibodies.This shows this
Inventive method fairly simple can be readily available more tumor specific antibodies, especially obtain the spy for not synantigen
Heterogenetic antibody, and tumour cell escape antibody can be thus reduced as far as possible for the antibody combined treatment of tumour in future
The probability for the treatment of.
Material and method
Material, cell and cell culture
HepG2 is purchased from American Type Culture Collecti (ATCC).Normal liver cell is purchased from ScienCell.DMEM culture mediums,
Fetal calf serum, mycillin, pancreatin/EDTA etc. are purchased from Thermo.Mouse monoclonal antibody hypotype identification kit, dimethyl are sub-
Sulfone (DMSO), paraformaldehyde, HAT additives, HT additives also have other chemical reagent to be purchased from Sigma.
HepG2 DMEM culture mediums (containing 10% fetal calf serum, adding 50IU/ml penicillin, 50 μ g/ml) cultures.Normally
Human liver cell (Cat.No.5200, ScienCell Research Laboratories, Inc.) hepatocyte cultures liquid
(Cat.No.5201, ScienCell Research Laboratories, Inc.) is cultivated.The culture environment of cell is, 37 DEG C,
5%CO2, humidity be more than 95%.
Human liver cell is immunized
When hepatocyte cultures are expanded to density and reach 80% or so, culture solution is gently sucked, is first washed away with PBS remaining
Then culture medium handles cell with PBS/10mM EDTA again, make cell detachment and finally piping and druming collection cell.Collect what is obtained
Cell, 1000 turns, after centrifugation in 4 minutes, cell dispels cell precipitation, and room temperature is fixed with 1% paraformaldehyde (being dissolved in PBS)
0.5 hour, paraformaldehyde is then washed away by centrifugation, and with PBS gravity treatment cells, after cell count, be finally stored in -20 DEG C.
1,000,000 cell constant volumes is taken (to be immunized use for the first time in 200 μ l PBS and with 200 μ l complete Freund's adjuvants or incomplete Freund's adjuvant
Complete Freund's adjuvant, after cannot be used up full Freund's adjuvant twice) fully vortex oscillation mixing, and pass through hypodermic injection respectively be immunized 6
Week old female BAl BIc/c mouse, 1,000,000 cellular immunities of every mouse, and 50 immune altogether, immunization interval 2 weeks, altogether three times.
Serum is collected in blood sampling after last time is 10 days immune.
Liver cancer cells are immunized
1, the amplification and pretreatment of liver cancer cells
When HepG2 cells are expanded to density and reach 80% or so, culture solution is gently sucked, first washes away remaining training with PBS
Base is supported, then handles cell with PBS/10mM EDTA again, makes cell detachment and finally piping and druming collection cell.Collect the thin of acquisition
Born of the same parents, 1000 turns, after centrifugation in 4 minutes, cell dispels cell precipitation, and room temperature fixes 0.5 with 1% paraformaldehyde (being dissolved in PBS)
Hour, paraformaldehyde is then washed away by centrifugation, and with PBS gravity treatment cells, after cell count, be finally stored in -20 DEG C.
2, the processing of mouse is immunized
56 week old female BAl BIcs/c mouse are taken, inject (tail vein injection or intraperitoneal injection) anti-HH cell respectively first
(injection is enough to dispose the anti-of HH cell and HepG2 common antigens for pure blood is clear or corresponding serum is purified polyclonal antibody
Serum or polyclonal antibody, the present embodiment have only used 0.3ml serum).During implementation steps 3, similary dosage will be used weekly
Serum processing, to maintain the concentration of anti-HH cell antibody enough in Mice Body.
3, HepG2 cells are immunized
After above-mentioned mouse injects anti-HH cell serum for the first time, immediately with the dosage of 1,000,000 HepG2 cells per mouses,
According to above-mentioned immunization method (identical with the method that human liver cell is immunized), these anti-HH cell serum of processing of subcutaneous inoculation
Processed mouse.It is important to note that as described in 2, (i.e. immunization interval is 2 during entire HepG2 cellular immunities
Be immunized altogether three times in week, for the first time with complete Freund's adjuvant, after cannot be used up full Freund's adjuvant twice, 6 weeks altogether), anti-HH
Cell serum need to be injected weekly once, each 0.3ml.After third time is immune after 2 weeks, strengthen exempting from 1,000,000 HepG2 intravenous injections
Epidemic disease, and the anti-HH cell serum of 0.3ml is injected simultaneously, mouse spleen and separating Morr. cell are taken after three days, is finally counted.
The acquisition of cell strain of monoclonal antibody
Then it is merged with 5: 1 cell proportion with SP 2/0, fusion agent PEG-1500.The cell merged averagely spread into
8 piece of 96 porocyte culture plates, (is contained with HAT culture mediums：100 μM of hypoxanthine, 0.4 μM of aminopterin, 16 μM of thymidine cores
Glycosides) selective killing culture is carried out, after about 10 days, the cell survived at this time is hybridoma.
The screening of cell strain of monoclonal antibody
HepG2 cell culture is in 96 porocyte culture plates, and when cell density reaches 90-100%, tenderness removes culture medium
And washed one time with PBS, 30 minutes then are fixed with PBS/1% paraformaldehyde room temperatures, is finally washed 3 times with PBS tendernesses.Then plus
Enter 100 μ l of monoclonal cell strain cells and supernatant, 37 DEG C, be incubated 1 hour.After tenderness washes away primary antibody, the sheep of HRP labels is added in
Anti-mouse secondary antibody, PBS/1%BSA dilutions, are incubated 1 hour by 37 DEG C.Finally tenderness washes away secondary antibody, and finally developed the color with TMB again
Liquid develops the color.At this time with the growing state of the result of TMB chromogenic reactions control Hybridoma Cell Culture plate, and determine to continue to select
Cell strain of monoclonal antibody.Then these cell strains are subcloned again, are subcloned required culture medium as HT culture mediums
(contain：100 μM of hypoxanthine, 16 μM of thymidines), the method for screening positive clone is as described above.Subclone screening has altogether
Three-wheel is carried out, and finally obtains stable cell strain of monoclonal antibody.
Monoclonal antibody hypotype is identified
Method according to the mouse monoclonal antibody hypotype kit of Sigma companies is identified.
Enzyme linked immunoassay (ELISA)
Cell (HH cell, HepG2 etc.) is incubated at 96 porocyte culture plates first, treats that cell density reaches 90-100%
When, tenderness removes culture medium and is washed one time with PBS, then fixes 30 minutes with PBS/1% paraformaldehyde room temperatures, finally uses
PBS tendernesses are washed 3 times.Then monoclonal cell strain cells and supernatant or its control are added in or with the diluted blood of PBS/1%BSA
Clear to wait samples, volume is all 100 μ l/ holes, 37 DEG C, is incubated 1 hour.After tenderness washes away primary antibody, the sheep anti-Mouse of HRP labels is added in
Secondary antibody, PBS/1%BSA dilutions, are incubated 1 hour by 37 DEG C.Finally tenderness washes away secondary antibody, and finally shown with TMB developing solutions again
Color.Entire reaction system is all 100 μ l.Negative control is SP2/0 cells and supernatants or PBS/1%BSA.
In the binding ability for detecting different cells, that is, the opposite recognition capability of antibody is detected, according to following calculation formula
It obtains：
With respect to the light absorption value of light absorption value/SP2/0 cells and supernatants of recognition capability=monoclonal antibody cells and supernatant.
In another embodiment of the invention, it provides a kind of common by removing cancer cell and corresponding normal cell in vitro
Antigen and the method for obtaining inhibiting tumor cell monoclonal antibody.
As shown in figure 5, for cancer cell and corresponding normal cell common antigen and most are removed described in the embodiment of the present invention in vitro
The schematic diagram of inhibiting tumor cell monoclonal antibody is obtained eventually.
The present embodiment method is equally immunized mouse with normal cell (such as HH cell) first and obtains anti-HH cell serum, and
Purifying obtains the polyclonal antibody of anti-HH cell, and the polyclonal antibody is coupled on agarose gel microsphere.Meanwhile this
Invention has cracked corresponding cancer cell (such as HepG2) and has obtained its lysate.Finally with the parent for being coupled anti-HH cell polyclonal antibodies
Remove part identical with HH cell in HepG2 cells with column.Last uncombined part is passed through as mice immunized with antigen
After being immunized three times, extracting spleen cell and with SP2/0 cell fusions, by screening, finally obtain the monoclonal antibody of anti-HepG2.
Specific method
The acquisition and coupling of anti-HH cell polyclonal antibodies
The preparation method of anti-HH cell serum is as previously mentioned, the antiserum obtained is coagulated with proteinA/G (GE) agarose
Glue microballoon is fully incubated, then with ice PBS fully wash until foreign protein completely clean, then delayed with 50mM acetate/acetics
Fliud flushing (PH3.0) comes out the antibody purification in serum, thus to obtain anti-HH cell polyclonal antibodies, and finally carries out dialysis PBS
In buffer solution.Finally, the specification method provided according to Bio-Rad companies is by antibody coupling to Affigel (Bio-Rad) pillar
On.
The acquisition of HepG2 antigens
HepG2 PBS/1%Triton X-100 add " cocktail " protease inhibitors (to contain：100nM Aprotinin,
50 μM of Leupeptin, 10 μM of PepstatinA, 0.2mM PMSF, 2mM Benzamidine) on ice crack 1 hour, 15000
It leaves the heart 10 minutes and obtains lysate supernatant.Then the lysate is added in anti-HH cell polyclonal antibody affinity columns,
And be fully incubated, the antigen thus shared in HepG2 cells with HH cell will be adsorbed by the affinity column, and HepG2 is specific
Antigen will not then combine the affinity column.Those remaining HepG2 lysates not combined with the affinity column finally carry out dialysis PBS
In buffer solution, thus to obtain HepG2 specific antigens, after determination of protein concentration, -20 DEG C of preservations are put.
It is prepared by monoclonal antibody
With the 6 week old BALB/c mouse of HepG2 specific antigens immunization Female of acquisition, immune dosage is 50 μ g every time,
It is 5 immune altogether, immunization interval 2 weeks, altogether three times.After last time is immune, extracting spleen cell obtains monoclonal with SP2/0 cell fusions
Cell strain.
The screening and identification of monoclonal antibody
It is identical with the method in previous embodiment.
As shown in fig. 6, identify HepG2 abilities for the monoclonal antibody specificity that in vitro method described in the embodiment of the present invention obtains
Column diagram.The results show that adopting said method, the present invention obtain 5 cell strain of monoclonal antibody, measured by hypotype altogether in figure,
Have 2 for IgG1 (PY1 and PY2), 3 are IGM (PY3, PY4 and PY5), in the contrast test with HH cell, find this 5
Kind antibody has HepG2 better recognition capability.
The method of the present invention can rapidly obtain the antibody of tumour-specific, and being quickly applied to oncotherapy for it provides
It may.For example, the present invention can obtain the tumour cell of patient and corresponding normal cell first, and fast in this way
Speed obtains the dedicated anti-tumour antibody of the patient, and using the treatment of its individuation.
Finally, the technology of the present invention can be applied not only to obtain the antibody of tumour-specific, can also distinguish other sections
Grind the cell of project research interested, such as terminal differentiation cell and stem cell.Other than distinguishing memebrane protein, it can also apply
In the differentiation of intracellular protein.
The above is only several embodiments of the present invention, any type of limitation is not done to the present invention, although this hair
It is bright to be disclosed as above with preferred embodiment, however not to limit the present invention, any person skilled in the art is not taking off
In the range of technical solution of the present invention, make a little variation using the technology contents of the disclosure above or modification is equal to
Case study on implementation is imitated, is belonged in technical solution of the present invention protection domain.
Claims (10)
- A kind of 1. easy method for finding cell-specific antigens, which is characterized in that include the following steps：A. anti-normal cell or the serum of normal structure are obtained；B. animal is handled with the step A antiserums obtained or the polyclonal antibody being purified；C. the processed animals of step B are immunized with corresponding cancer cell or cancerous issue；D. the B cell of animal after separating step C is handled；E. the B cell after detaching is merged with myeloma cell, obtains cell strain of monoclonal antibody；With cell strain of monoclonal antibody system Standby monoclonal antibody；F. the cell pyrolysis liquid for the monoclonal antibody affinity purification cancer cell that step E is obtained, obtains antigen protein in connection.
- 2. the method for unknown specific antigen cancer cell specific antibody is obtained according to claim 1, which is characterized in that institute Cancer cell is stated as liver cancer cells.
- 3. the method for unknown specific antigen cancer cell specific antibody is obtained according to claim 1, which is characterized in that into One step includes：G. the antigen protein that mass spectrometry procedure F is obtained.
- 4. the method for unknown specific antigen cancer cell specific antibody is obtained according to claim 1, which is characterized in that institute Step A is stated to further comprise：A1. cell is collected；A2. gravity treatment cell；A3. serum is obtained after being immunized.
- 5. the method for unknown specific antigen cancer cell specific antibody is obtained according to claim 4, which is characterized in that institute Sub-step A1 is stated to further comprise：When normal cell culture is expanded to density and reaches 75~85%, culture solution is sucked；Remaining culture medium first is washed away with PBS, Then cell is handled with PBS/8-12mM EDTA again, makes cell detachment and finally piping and druming collection cell.
- 6. the method for unknown specific antigen cancer cell specific antibody is obtained according to claim 4, which is characterized in that institute Sub-step A2 is stated to further comprise：800~1200 turns, after centrifugation in 3-5 minutes, cell precipitation is dispelled with 1% paraformaldehyde, and to fix 0.3~0.7 small for room temperature When, paraformaldehyde is then washed away by centrifugation, and with PBS gravity treatment cells, after cell count, be finally stored in -20 DEG C.
- 7. the method for unknown specific antigen cancer cell specific antibody is obtained according to claim 4, which is characterized in that institute Sub-step A3 is stated to further comprise：1,000,000 cell constant volumes is taken to be mixed in 200 μ l PBS and with 200 μ l complete Freund's adjuvants or incomplete Freund's adjuvant vortex oscillation It closes；Immune 5-7 week old mouse, 1,000,000 cellular immunities of every mouse, immunization interval 2 weeks are 2-4 times common；The immune 5- of last time Serum is collected in blood sampling after 15 days.
- 8. the method for unknown specific antigen cancer cell specific antibody is obtained according to claim 1, which is characterized in that institute Step C is stated to further comprise：C1. cell is collected；C2. gravity treatment cell；C3. B cell is obtained after being immunized.
- 9. the method for unknown specific antigen cancer cell specific antibody is obtained according to claim 8, which is characterized in that institute Sub-step B1 is stated to further comprise：When cancer cell culture is expanded to density and reaches 78-82% or so, culture solution is sucked；First use PBS Remaining culture medium is washed away, then handles cell with PBS/7-13mM EDTA again, makes cell detachment and finally blow and beat to collect carefully Born of the same parents.
- 10. the method for unknown specific antigen cancer cell specific antibody is obtained according to claim 8, which is characterized in that The sub-step B2 further comprises：900~1100 turns, after centrifugation in 3-5 minutes, cell precipitation is dispelled with 0.5-1.5% paraformaldehydes, and room temperature fix 0.3~ 0.7 hour, paraformaldehyde is then washed away by centrifugation, and with PBS gravity treatment cells, after cell count, be finally stored in -20 DEG C.
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| PCT/CN2018/093283 WO2019144569A1 (en) | 2018-01-26 | 2018-06-28 | Method for screening specific antibodies against different antigens |
| PCT/CN2018/093245 WO2019144568A1 (en) | 2018-01-26 | 2018-06-28 | Highly effective anti-cancer cell monoclonal antibody screening method |
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| WO2019144568A1 (en) * | 2018-01-26 | 2019-08-01 | 裴新辉 | Highly effective anti-cancer cell monoclonal antibody screening method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1786298A (en) * | 2005-10-28 | 2006-06-14 | 上海博昂抗体生物科技有限公司 | Preparation method of anticell or tissue protein mono clone antibody stock |
| US20070292428A1 (en) * | 2004-07-10 | 2007-12-20 | Alexion Pharmaceuticals, Inc. | Antibodies Against Cancer Produced Using Masked Cancer Cells as Immunogen |
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2018
- 2018-01-26 CN CN201810081245.XA patent/CN108250287A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070292428A1 (en) * | 2004-07-10 | 2007-12-20 | Alexion Pharmaceuticals, Inc. | Antibodies Against Cancer Produced Using Masked Cancer Cells as Immunogen |
| CN1786298A (en) * | 2005-10-28 | 2006-06-14 | 上海博昂抗体生物科技有限公司 | Preparation method of anticell or tissue protein mono clone antibody stock |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019144568A1 (en) * | 2018-01-26 | 2019-08-01 | 裴新辉 | Highly effective anti-cancer cell monoclonal antibody screening method |
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