CN103073644A - Specific anti-mouse TIGIT monoclonal antibody and preparation method, identification and application thereof - Google Patents
Specific anti-mouse TIGIT monoclonal antibody and preparation method, identification and application thereof Download PDFInfo
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Abstract
The invention relates to an anti-mouse TIGIT (T cell Ig and ITIM domain), and a hybridoma cell strain mTIGIT-mAb-13G6 (the preservation NO: CCTCC NO: C201299) for producing the monoclonal antibody, and further relates to a preparation method and the application of the monoclonal antibody. The monoclonal antibody can efficiently prevent combination of mTIGIT and mPVR (poliovirus receptor), and can be used for Western blot, ELISA and Flow Cytometry for mTIGIT molecule detection.
Description
Technical field
The present invention relates to the monoclonal antibody of the anti-mouse TIGIT of a strain (full name T cell Ig and ITIM domain).Specifically, relating to has specific monoclonal antibody to TIGIT, the invention still further relates to the hybridoma cell strain that produces this monoclonal antibody, and the preparation method of described monoclonal antibody and application.
Background technology
Natural killer cell (namely, the NK cell) as main innate immune cells, kill and wound at body and to play an important role aspect virus infected cell and the tumour cell, the NK cell is by the different activated form acceptor in its surface and the function such as suppress that receptor is controlled the activation of NK cell, breeds and killed and wounded.
TIGIT (T cell Ig and ITIM domain) molecule is that the inhibition acceptor at the NK cell surface is expressed in new discovery in 2009, mainly comprise the outer IgV-spline structure territory of born of the same parents, ITIM motif (Immunoreceptor tyrosine-based inhibitory motif) in cross-film district and the born of the same parents, with high-affinity binding partner PVR (poliovirus receptor), and by ITIM motif transmission Inhibitory signal in the born of the same parents, thereby the killing ability that suppresses the NK cell is with activation and the function of reactivity acceptor CD226/CD96 coordinated regulation NK cell.
In the melanotic tumor tumor-bearing mice, TIGIT up-regulated, tumour cell be high expression level part PVR again, and TIGIT in conjunction with the ability of PVR far above CD226/CD96, this can cause the tumour immunity tolerance so that the NK cytoactive is suppressed, and causes tumour immunity to be escaped.
Eukaryotic expression mTIGIT-hFc fusion rotein, immune rat, get splenocyte and the myelomatosis IR983F cytogamy of the rear rat of immunity, filter out and to secrete the hybridoma of the monoclonal antibody that mTIGIT is responded, and its antibody capable is efficiently blocked the combination of mTIGIT and part mPVR, obtains monoclonal antibody from the hybridoma culture supernatant or from the nude mice ascites of intraperitoneal inoculation hybridoma.
Preparation mTIGIT barrier monoclonal antibody, for the detection of mTIGIT molecule and the research of mTIGIT function provide the foundation, this antibody can be widely used in the research of molecular immunology, and can be used as the potential treatment tool of breaking the tumour immunity tolerance.
Summary of the invention
An object of the present invention is to provide has specific monoclonal antibody to mouse TIGIT (mTIGIT).
A further object of the present invention provides the hybridoma cell strain of the monoclonal antibody that produces the anti-mouse TIGIT of specificity.
Another purpose of the present invention provides the preparation method of monoclonal antibody of the present invention.
Another purpose of the present invention provides monoclonal antibody of the present invention for detection of the application of mTIGIT, described monoclonal antibody can be blocked the combination of mTIGIT and its part PVR, can detect mTIGIT by being used for Western blot, ELISA or Flow Cytometry.
One aspect of the present invention relate to a kind of to mTIGIT have specific monoclonal antibody with and preparation method thereof, the type of described antibody is IgG2a, κ, and can efficiently block the combination of mTIGIT and mPVR.
Another aspect of the present invention relates to the hybridoma cell strain of a strain stably excreting mTIGIT monoclonal antibody, its called after mTIGIT-mAb-13G6, deposit number is CCTCC NO:C201299, be preserved in (address: Wuhan, China, Chinese Typical Representative culture collection center on September 25th, 2012, Wuhan University, postcode: 430072).
Another aspect of the present invention relate to screening can expression specificity in conjunction with the method for the hybridoma cell strain of the monoclonal antibody of the anti-mouse TIGIT of mouse TIGIT, described method comprises: the expression and purification of the fusion rotein mTIGIT-hFc of mTIGIT and people Fc, use the mTIGIT-hFc immune rat, get splenocyte and the myelomatosis IR983F cytogamy of the rear rat of immunity, the screening hybridoma, the high affine mTIGIT-hFc of the monoclonal anti physical efficiency specificity of its secretion, with the cell strain called after mTIGIT-mAb-13G6 that screening obtains, deposit number is CCTCC NO:C201299.
Efficiently blocked the combination of mTIGIT and mPVR by the anti-mouse TIGIT monoclonal anti physical efficiency of cell strain mTIGIT-mAb-13G6 secretion.
In addition, anti-mouse TIGIT monoclonal antibody of the present invention is also referred to as the 13G6 monoclonal antibody.
A preferred aspect of the present invention relates to the method for preparing anti-mouse TIGIT monoclonal antibody, and described method comprises from preserving number and is the hybridoma cell strain mTIGIT-mAb-13G6 culture supernatant of CCTCC NO:C201299 or obtains described monoclonal antibody from the nude mice ascites of the described hybridoma cell strain of intraperitoneal inoculation.
Another aspect of the present invention relates to monoclonal antibody to the blocking effect of mTIGIT and mPVR combination.Detect blocking-up efficient with monoclonal antibody of the present invention, the result shows that the monoclonal anti physical efficiency is blocked the combination of mTIGIT and mPVR efficiently.
The monoclonal antibody that relates to another aspect of the present invention detects among the mTIGIT at Western blot and uses.With Identification of Monoclonal Antibodies mTIGIT protein expression of the present invention, Western blot tests demonstration: the monoclonal anti physical efficiency in Western blot level for detection of the mTIGIT-hFc fusion rotein.
Another aspect of the present invention relates to monoclonal antibody in the application of ELISA level detection mTIGIT.With the mTIGIT-hFc of monoclonal antibody detection different concns of the present invention, the result shows: the monoclonal anti physical efficiency is used for ELISA and detects mTIGIT-hFc albumen.
Another aspect of the present invention relates to the monoclonal anti physical efficiency and is used for Flow Cytometry detection mTIGIT.Can be used for mark mTIGIT positive cell with labeling of monoclonal antibodies of the present invention, and mark mTIGIT negative cells not.Show that monoclonal anti physical efficiency of the present invention is used for the expression that Flow Cytometry detects mTIGIT.
The monoclonal antibody that the invention still further relates to anti-mouse TIGIT of the present invention is for the preparation of the application in the test kit that detects mTIGIT, and described test kit utilizes the monoclonal antibody of anti-mouse TIGIT of the present invention to detect mTIGIT by Western blot, ELISA or Flow Cytometry.
The invention still further relates to the test kit of a kind of mTIGIT of detection, described test kit comprises the monoclonal antibody of anti-mouse TIGIT of the present invention.
The invention still further relates to the method for a kind of mTIGIT of detection, described method comprises the steps: that (1) hatch monoclonal antibody of the present invention with the cell that separates to be detected or the lysate of this cell, and (2) have judged whether positive reaction.Wherein said positive reaction refers to the combination of mTIGIT in the anti-mTIGIT antibody of specificity of the present invention and the testing sample, and described positive reaction can be judged by the immune marking, ELISA or Flow cytometry.
Among the present invention, term " specificity of monoclonal antibody " refers to defined epitope on the monoclonal antibody identification antigen or antigenic determinant and the character of combination with it.
Term " reactivity of monoclonal antibody " refers under suitable reaction conditions the ability that monoclonal antibody is combined with antigen.
Term " cell strain " refers to by screening or limiting dilution method, from the single cell culture thing of primary culture or clone acquisition.
According to of the present invention open, select the mTIGIT-hFc fusion rotein, being prepared with specific monoclonal antibody according to method described herein is apparent to those skilled in the art, should be considered as within the scope of the present invention.
In sum, beneficial effect of the present invention is: the invention provides preparation method, evaluation and the application of the monoclonal antibody of a kind of anti-mouse TIGIT, this monoclonal antibody has the height of tiring, the advantages such as specificity is good, and can efficiently block the combination of mTIGIT and mPVR, be applicable to the detection of different specimens.Monoclonal antibody provided by the invention can be widely used in the means of different such as Western blot, ELISA, flowCytometry and detect mTIGIT albumen, for the function of studying mTIGIT provides the foundation.
The implication of shortenings:
MTIGIT: mouse TIGIT (T cell Ig and ITIM domain)
MPVR: mouse PVR (poliovirus receptor), the part of mouse TIGIT
Description of drawings
From the detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1, the structure of carrier pcDNA3-mTIGIT-hFc.
Fig. 2, mTIGIT-hFc expressing fusion protein, purifying and evaluation, wherein:
Fig. 2 A, the qualification result of mTIGIT-hFc fusion protein purification;
Fig. 2 B, the Western blot qualification result of mTIGIT-hFc fusion rotein;
Fig. 3, monoclonal antibody Purity result of the present invention.
Fig. 4, monoclonal antibody specificity qualification result of the present invention, wherein:
Fig. 4 A, Flow cytometry method identifies that monoclonal antibody is to the specific result of mTIGIT;
1) the 293T-mTIGIT/hTIGIT/mCD226/mCD96 positive rate detects
2) the hTIGIT/CD226/CD96 molecule of monoclonal antibody nonrecognition cell surface of the present invention
Fig. 4 B, indirect elisa method checking monoclonal antibody is to the specific result of mTIGIT;
Fig. 4 C, Western blot method identifies that monoclonal antibody is to the specific result of mTIGIT.
Fig. 5, the qualification result of monoclonal antibody block function of the present invention, wherein:
Fig. 5 A, the 293T-mPVR transfection efficiency detects;
Fig. 5 B, monoclonal antibody of the present invention is efficiently blocked the combination of 293T-mPVR and mTIGIT-hFc albumen;
Fig. 6, monoclonal antibody of the present invention is used for the result that Western blot detects mTIGIT.
Fig. 7, monoclonal antibody of the present invention is used for the result that ELISA detects mTIGIT.
Fig. 8, monoclonal antibody of the present invention is used for the result that FACS detects mTIGIT.
A. monoclonal antibody of the present invention detects the mTIGIT of 293A-mTIGIT cell surface;
B. commercial streaming antibody (Anti-Mouse TIGIT Alexa
647, eBioscience, Cat.NO.50-9501) detect the mTIGIT of 293A-mTIGIT cell surface.
The preservation explanation
The Rat hybridoma cell strain mTIGIT-mAb-13G6 of the monoclonal antibody of the anti-mouse TIGIT of secretion specificity is preserved in (address: Wuhan, China, Chinese Typical Representative culture collection center on September 25th, 2012, Wuhan University, postcode: 430072), deposit number is CCTCC NO:C201299.
The sequence table explanation
Embodiment
Following examples will help those of ordinary skill in the art further to understand the present invention, but not limit in any form the present invention.
Experimental technique among the embodiment if no special instructions, all adopts this area routine techniques, and experiment reagent is the commercially available prod.
Embodiment 1: the structure of carrier for expression of eukaryon pcDNA3-mTIGIT-hFc
(pMD18-T is available from Takara from pMD18-T-total length mouse TIGIT carrier, Code:D103A) pcr amplification mouse TIGIT extracellular fragment encoding sequence in, (pcDNA3 is available from Invitrogen with sequence fragment and pcDNA3-hFc recombinant plasmid, numbering V790-20) EcoRI with connect after Xho I place enzyme is cut, connect product and transform DH5 α intestinal bacteria, the coated plate grow overnight, and the bacterial strain that screening obtains is carried out PCR detect, PCR is identified that positive clone carries out dna sequencing, and sequencing result shows that carrier pcDNA3-mTIGIT-hFc successfully constructs.The structure flow process of carrier for expression of eukaryon pcDNA3-mTIGIT-hFc as shown in Figure 1.
Embodiment 2: the expression of fusion rotein mTIGIT-hFc, purifying and evaluation
1, the expression of fusion rotein mTIGIT-hFc (SEQ ID NO:3)
With pcDNA3-mTIGIT-hFc carrier transfection 293A cell (available from the Shanghai cell bank, catalog number (Cat.No.): GNHu 1), collect serum-free culture thing supernatant, 30kD film (available from the green skies) concentrates supernatant, with Protein G purification column (available from GE Healthcare, HiTrap Protein G HP, article No.: 1700404-01) concentrated supernatant is carried out the enrichment of target protein, then use acid PBS (pH2.8) to carry out wash-out, again the protein solution of wash-out neutralized with alkaline Tris damping fluid (pH9.0), namely obtain the mTIGIT-hFc protein solution.The result is shown in Fig. 2 A.
2, the evaluation of fusion rotein
The mTIGIT-hFc albumen of purifying is carried out SDS-PAGE﹠amp; Western Blot identifies; Result such as Fig. 2 B.
Embodiment 3: the preparation of monoclonal antibody
1, the preparation of splenocyte
The mTIGIT-hFc albumen of purifying and equal-volume complete Freund's adjuvant (CFA, available from Sigma, article No. F5881-10ML) mixes, the final concentration of mTIGIT-hFc is 100 μ g/ml, and the abundant mixing of mixture is formed water-in-oil, gets rat (available from Shanghai Si Laike, male, 8 ages in week) carry out initial immunity, every rat injection 40-60 μ g mTIGIT-hFc albumen (immunizing dose is 133-200 μ g/kg rat body weight), interval 2 all immunity are once.After the immunity 5 times, the serum titer that enzyme-linked immunosorbent assay (ELISA) detects rat is very high, gets Rats Spleen after rat is put to death, and crosses 200 order cells sieve, collects the splenocyte that filters, and after leaving standstill several minutes, draws the upper strata splenocyte.
The method that enzyme-linked immunosorbent assay (ELISA) detects antibody titer is as follows: with albumen to the 10 μ g/ml of coating buffer (0.1M carbonate buffer solution pH 9.6) dilution purifying, 100 μ l/ holes are coated with 96 hole elisa plates, 4 ℃ are spent the night, clean 3 times with PBST (0.05%Tween20-PBS pH 7.4), the 1%BSA sealing was hatched 2 hours for 37 ℃.PBST cleans 3 times, adds the rat blood serum (experimental group) of doubling dilution to be measured, and 6-7 gradient is set, and not immune healthy rat serum is done negative control, and hatched 1 hour for 37 ℃ in 100 μ l/ holes.PBST cleans 3 times, and every hole adds the sheep anti-mouse antibody (available from Boster company, article No. BA1050) of horseradish peroxidase (HRP) mark of 100 μ l dilution in 1: 10000, hatches 1 hour for 37 ℃.Add tmb substrate liquid (available from eBioscience company, article No. 00-4201-56) after cleaning, 100 μ l/ holes, lucifuge colour developing 15 minutes adds stop buffer (2MH
2SO
4) 50 μ l/ holes, detect immediately OD490 after the termination.
2, the preparation of feeder cell
In cytogamy the day before yesterday, the preparation rat peritoneal macrophages is as feeder layer cells.Concrete grammar is as follows: normal rat dislocation in 8 ages in week is put to death, and 75% alcohol disinfecting exposes belly, cuts off peritonaeum.Draw the incomplete nutrient solution of 5ml DMEM (available from Gibco company) with the 10ml syringe and inject mouse peritoneal, suction for several times repeatedly.With syringe pumpback intraperitoneal liquid, inject centrifuge tube.With the incomplete substratum washing of DMEM 2 times, 4 ℃ of centrifugal 10min of 300g abandon supernatant.With DMEM (containing 10% calf serum) nutrient solution re-suspended cell, adjusting cell concn is 2 * 10
5/ ml adds 100 and enters in 96 holes, and every hole 100 μ l put into cell culture incubator, and 37 ℃, 5%CO
2Cultivate under the condition.
3, the preparation of cytogamy and hybridoma
Get splenocyte (10-15 * 10 that prepare
7) and myeloma cell IR983F (available from logical (Shanghai) bio tech ltd, 5 * 10 of sending
7) abundant mixing in serum-free DMEM substratum, centrifugal afterwash substratum, the cell of upspringing gently, PEG (molecular weight 1,000-6, the 000) solution that adds preheating, the final concentration of PEG is 50% (W/V), add serum-free DMEM substratum (available from Gibco company) after 60 seconds, the centrifugal afterwards cell of collecting precipitation adds HAT and selects substratum resuspended.HAT selects substratum for comprising the DMEM substratum of 20%FCS (calf serum), 10mM sodiumhypoxanthine (xanthoglobulin), 40mM aminopterin (aminopterin), 1.6mMthymidine (thymus pyrimidine).
The cell suspension that merges is added to previously prepared feeder cell (namely, the feeder cell of preparation in above-mentioned 2) in, fused cell selects culture medium culturing after one week at HAT, use HT substratum (the DMEM substratum that comprises 20%FCS, 10mM sodium hypoxanthine, 1.6mM thymidine) instead and continue to cultivate time enough, then screening produces the hybridoma of purpose antibody and carries out ELISA and detect.Replacing the HT nutrient solution with the DMEM nutrient solution that contains 10% calf serum simultaneously cultivates.
Adopt the limiting dilution method to carry out subcloning the hybridoma of the generation monoclonal antibody of the present invention that obtains, obtain hybridoma cell strain mTIGIT-mAb-13G6 after many screenings of ELISA, vitro culture is more than 2 months or after frozen 6 months continuously, and the antibody of anti-mouse TIGIT still can be stablized and be secreted in a large number to this cell strain.This hybridoma cell strain on September 25th, 2012 be preserved in Chinese Typical Representative culture collection center (address: Wuhan, China, Wuhan University, postcode: 430072), deposit number is CCTCC NO:C201299.
3, the preparation of monoclonal antibody
During the preparation monoclonal antibody, one can obtain antibody from above-mentioned Hybridoma Cell Culture supernatant, and specifically, passage is cultivated after 2 days and received culture supernatant, contains the monoclonal antibody of higher concentration in the supernatant.They are two years old, described hybridoma can be expelled to the nude mice abdominal cavity with the paraffin preimmunization, extract the acquisition monoclonal antibody by extracting nude mice ascites, specifically, the female nude mice in paraffin immunity 8-10 age in week, every nude mice abdominal cavity is injected the aseptic whiteruss of 500 μ l; Abdominal injection hybridoma after one week, every nude mice injection 1-2 * 10
6Cell produced ascites about 7-10 days, collected nude mice ascites, and centrifugal receipts supernatant is ascites antibody.By the antibody that aforesaid method obtains, can use Protein G affinity chromatography method purifying, the purity of antibody is identified with SDS-PAGE.As shown in Figure 3, the purity of purified monoclonal antibody is near 100%, the about 140kDa of monoclonal antibody molecule amount, the about 45kDa of heavy chain wherein, the about 25kDa of light chain.
Embodiment 4: the evaluation of monoclonal antibody
1, indirect ELISA is measured antibody titer
The method that ELISA detects antibody titer is as follows:
With 0.1M carbonic acid buffer dilution mTIGIT-hFc albumen to 0.25 μ g/ml, 100 μ l/ holes, coated 96 hole elisa plates, 4 ℃ are spent the night, and clean 3 times with PBST (0.05%Tween20-PBS, pH 7.4), and the 1%BSA sealing was hatched 2 hours for 37 ℃.PBST cleans 3 times, add hybridoma mTIGIT-mAb-13G6 culture supernatant or ascites through doubling dilution, 8-12 gradient is set, with other hybridoma (for example, pK136 is available from Chinese Academy of Sciences's Shanghai cell bank) culture supernatant or ascites works as negative control, and establish PBS and be the zeroing hole, hatched 1 hour for 37 ℃ in 100 μ l/ holes.PBST cleans 3 times, and every hole adds the mouse IgG of the Chinese People's Anti-Japanese Military and Political College (OLIGOAB02H) antibody (dilution in 1: 2000, this antibody is available from Absea) of 100 μ l horseradish peroxidase (HRP) marks, hatches 1 hour for 37 ℃.Add tmb substrate liquid (available from eBioscience company, article No. 00-4201-56) after cleaning, 100 μ l/ holes, lucifuge colour developing 10-15 minute adds stop buffer (2M H
2SO
4) 100 μ l/ holes, microplate reader detects OD450 and OD630 after stopping immediately.Compare with the PBS hole, when test antibody detected value/negative control value (ratio) is made as the positive greater than 2.1, take in the positive hole of greatest dilution as the titre of antibody.
2, BIAcore 3000 measures the affinity costant of monoclonal antibody.
Put into the CM5 chip, Dock makes moving phase with PBS, twice of sensitization.Inject first 50mMNaOH+0.05%SDS (10 μ l/min, 2min), then inject 50mM NaOH (10 μ l/min, 2min) flushing chip surface.At first grope the mTIGIT-hFc concentration and the pH that suit, final defined antigen concentration is 10 μ g/ml, pH=5.0.
Injection activation solution (sodium-acetate of pH=5.5 and pH=5.0 was by 1: 1 volume ratio mixing) (5 μ l/min, 7min,) the activation chip, with the NaAc of PH 5.0 with antigen (namely, mTIGIT-hFc) be diluted to 10 μ g/ml, the antigen of injection dilution begins to carry out antigenic mark, passage of mark stays another passage to do blank.With the thanomin sealing, off-period is identical when flow velocity is with activation.1 * PBS sensitization twice with different antibodies concentration determination mark effect, is groped regeneration condition simultaneously, can determine that 50mM NaOH is proper regeneration condition.With the 13G6 monoclonal antibody (namely, anti-mouse TIGIT monoclonal antibody of the present invention) with 1 * PBS doubling dilution to 0.24 μ g/ml, 0.08 μ g/ml, 0.0267 μ g/ml, 0.0089 μ g/ml, 0.00296 μ g/ml, 0.0001 μ g/ml, 0.000033 seven concentration of μ g/ml, setting program are measured the 13G6 monoclonal antibody to the avidity of antigen mTIGIT-hFc.Obtain carrying out match behind the avidity curve, draw affinity constant K, the L/M of unit sees the following form 1.
3, the Ig subclass of monoclonal antibody is measured
Subclass, the culture supernatant of monoclonal antibody tired, ascites antibody is tired and affinity costant the results are shown in Table 1.
Subclass, the culture supernatant of table 1. monoclonal antibody of the present invention tired, ascites antibody is tired and affinity costant
| Subclass | IgG2a，κ |
| The culture |
10 -4～10 -3 |
| Ascites antibody is tired | 1×10 -12 |
| Affinity costant (L/M) | 7.14×10 11 |
4, the specificity identification of monoclonal antibody
HTIGIT, mCD226 and mCD96 molecule and mTIGIT numberator height homology, take 13G6mAb as primary antibodie, respectively with cell 293T-mTIGIT, 293T-hTIGIT, 293T-mCD226 and 293T-mCD96 are in conjunction with (above-mentioned four kinds of cells are prepared according to ordinary method by the inventor: make up recombinant vectors pcDNA3-mTIGIT, pcDNA3-hTIGIT, pcDNA3-mCD226 and pcDNA3-mCD96, with the recombinant vectors difference transfection 293T cell (the 293T cell is available from the Shanghai cell bank) that makes up), use again the mouse IgG2a of the PE-Chinese People's Anti-Japanese Military and Political College (available from eBioscience, article No. 12-4817) is two anti-detection 13G6 monoclonal antibodies, the result is shown in Fig. 4 A, 13G6 only can specific recognition mTIGIT molecule, its excess-three kind molecule without identification, is illustrated that 13G6mAb is the monoclonal antibody specific for mTIGIT.
With the specificity of indirect elisa method checking monoclonal antibody, 13G6mAb is primary antibodie, and the mouse IgG of the Chinese People's Anti-Japanese Military and Political College (OLIGOAB02H) (available from Absea) of HRP mark detects as the two anti-ELISA that carry out, and the basic debond eGFP-hFc of 13G6mAb is shown in Fig. 4 B.
Adopt the method for immunoblotting to carry out specific detection, we with this monoclonal antibody as primary antibodie, the mouse IgG of the Chinese People's Anti-Japanese Military and Political College (OLIGOAB02H) (available from Absea) of HRP mark detects as the two anti-Western blot that carry out, detect fusion rotein mTIGIT-hFc and eGFP-hFc, identify through Western blot, shown in Fig. 4 C, located a specific band at molecular weight 41kDa (mTIGIT-hFc), and (eGFP-hFc) locates there is not specific band about 55kDa.Proving that further the secreted monoclonal antibody of hybridoma is reactionless to hFc, is the monoclonal antibody specific for mTIGIT.5, the evaluation of monoclonal antibody block function
The 13G6mAb cell culture supernatant is hatched with 1 μ g mTIGIT-hFc albumen first, and 4 ℃, 20min; Use again the resuspended 293T-mPVR cell of mixtures incubated (the 293T cell is available from the Shanghai cell bank, catalog number (Cat.No.): GNHu17), 4 ℃, 20min; PBS cleans 3 times, the centrifugal 5min of 3500rpm, and the anti-human Fc of mark streaming antibody PE-goat (available from BD Bioscience), 4 ℃, 20min changes the streaming pipe over to, detects (BD FACSCalibur) with small-sized flow cytometer and detects.The result is shown in Fig. 5 B, and contrast is complete to be blocked and not blocking-up group, and 13G6mAb can block the combination of mTIGIT and mPVR substantially fully, illustrates that this monoclonal antibody has efficient blocking ability.Embodiment 5: the application of anti-mouse TIGIT monoclonal antibody
1) is used for Western blot and detects mTIGIT
With the monoclonal antibody of purifying as primary antibodie, the rabbit Chinese People's Anti-Japanese Military and Political College murine antibody of HR mark is (available from BOSTER, article No.: BA1058) detect as the two anti-Western blot that carry out, such as Fig. 6, this monoclonal antibody can detect the mTIGIT-hFc albumen in the full lysate of 293A-mTIGIT-hFc cell, does not detect signal in the 293A-eGFP-hFc total protein.
2) be used for ELISA and detect mTIGIT
Respectively with coated 96 orifice plates of the fusion rotein mTIGIT-hFc of different concns, prepared monoclonal antibody is as primary antibodie, the mouse IgG of the Chinese People's Anti-Japanese Military and Political College (OLIGOAB02H) (available from Absea) of HRP mark is anti-as two, add at last the TMB nitrite ion (available from eBioscience, article No. 00-4201-56), detects OD450 and OD630, calculate Δ 450=OD450-OD630, do Δ 450 graphic representations corresponding to fusion rotein mTIGIT-hFc of different concns, R after over-fitting
2Can reach the ELISA requirement.The results are shown in such as Fig. 7, show that this monoclonal antibody can be used in ELISA and detects mTIGIT albumen.
3) be used for Flow Cytometry and detect mTIGIT
FACS detects mTIGIT at clone 293A-mTIGIT (pcNDA3-mTIGIT plasmid transfection 293A cell, 293A is available from the Shanghai cell bank) and the expression on 293A surface, prepared monoclonal antibody is as primary antibodie, anti-as two with the mouse IgG2a of the PE-Chinese People's Anti-Japanese Military and Political College (available from BD Bioscience), positive cell (293A-mTIGIT) and negative cells (293A) with monoclonal antibody 13G6 marker expression mTIGIT of the present invention, shown in Fig. 8 (A), prepared monoclonal antibody can be incorporated into the 293A-mTIGIT cell surface, but not with the 293A Cell binding.Contrast commercial Alexa 647/mTIGIT mark mTIGIT, such as Fig. 8 (B), both ratios approach, and the result shows that monoclonal antibody of the present invention can be used for flow cytometer detection mouse TIGIT molecule.
Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and describe, but will be understood by those skilled in the art that, under the condition that does not deviate from by the spirit and scope of the present invention as defined in the claims, the variation of various forms and details can be carried out therein, the arbitrary combination of various embodiments can be carried out.
Claims (9)
1. the monoclonal antibody of the anti-mouse TIGIT of a species specificity, described antibody are that the Rat hybridoma cell strain mTIGIT-mAb-13G6 secretion of CCTCC NO:C201299 produces by preserving number.
2. according to antibody claimed in claim 1, its hypotype is IgG2a, κ.
3. according to antibody claimed in claim 1, it can efficiently block the combination of mTIGIT and mPVR.
4. produce the hybridoma cell strain mTIGIT-mAb-13G6 of the described monoclonal antibody of claim 1, its preserving number is CCTCC NO:C201299.
5. the method for preparing monoclonal antibody claimed in claim 1, described method comprise from preserving number and are the hybridoma cell strain mTIGIT-mAb-13G6 culture supernatant of CCTCC C201299 or obtain described monoclonal antibody from the nude mice ascites of the described hybridoma cell strain of intraperitoneal inoculation.
6. the monoclonal antibody of claim 1 is for the preparation of the application in the test kit that detects mTIGIT, and described test kit utilizes the monoclonal antibody of claim 1 to detect mTIGIT by Western blot, ELISA or Flow Cytometry.
7. test kit that detects mTIGIT, described test kit comprises the monoclonal antibody of anti-mouse TIGIT claimed in claim 1.
8. method that detects mTIGIT, described method comprises the steps:
(1) monoclonal antibody claimed in claim 1 is hatched with the cell that separates to be detected or the lysate of this cell;
(2) judged whether positive reaction.
9. according to claim 8 method, wherein said positive reaction is judged by the immune marking, ELISA or Flow cytometry.
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