CN103073644B - 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
本发明涉及一种抗小鼠TIGIT的单克隆抗体，本发明还涉及生产该单克隆抗体的杂交瘤细胞株mTIGIT-mAb-13G6(保藏编号：CCTCC NO：C201299)，本发明还涉及该单克隆抗体的制备方法及应用，本发明的单克隆抗体能高效阻断mTIGIT与mPVR的结合，可以用于Western blot、ELISA、Flow Cytometry检测mTIGIT分子。The present invention relates to an anti-mouse TIGIT monoclonal antibody. The present invention also relates to the hybridoma cell line mTIGIT-mAb-13G6 (preservation number: CCTCC NO: C201299) producing the monoclonal antibody. The present invention also relates to the monoclonal antibody The preparation method and application of the antibody, the monoclonal antibody of the present invention can efficiently block the combination of mTIGIT and mPVR, and can be used to detect mTIGIT molecules by Western blot, ELISA, and Flow Cytometry.
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, relate to TIGIT is had to specific monoclonal antibody, 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 (, NK cell) as main innate immune cells, at body, kill and wound aspect virus infected cell and tumour cell and play an important role, 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 NK cell surface is expressed in new discovery in 2009, mainly comprise the outer IgV-spline structure of born of the same parents territory, ITIM motif (Immunoreceptor tyrosine-based inhibitory motif) in cross-film district and born of the same parents, with high-affinity binding partner PVR (poliovirus receptor), and transmit Inhibitory signal by ITIM motif in born of the same parents, thereby the killing ability that suppresses NK cell, with activation and the function of reactivity acceptor CD226/CD96 coordinated regulation NK cell.
In melanotic tumor tumor-bearing mice, TIGIT up-regulated, tumour cell is high expression level part PVR again, and TIGIT in conjunction with the ability of PVR far above CD226/CD96, this is suppressed NK cytoactive, can cause tumour immunity tolerance, 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 can 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, from hybridoma culture supernatant or from the nude mice ascites of intraperitoneal inoculation hybridoma, obtain monoclonal antibody.
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 tumour immunity tolerance.
Summary of the invention
An object of the present invention is to provide mouse TIGIT (mTIGIT) is had to specific monoclonal antibody.
A further object of the present invention is to provide the hybridoma cell strain of the monoclonal antibody that produces the anti-mouse TIGIT of specificity.
Another object of the present invention is to provide the preparation method of monoclonal antibody of the present invention.
Another object of the present invention is to provide 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 be by detecting mTIGIT 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, on September 25th, 2012, be preserved in (address: Wuhan, China, Chinese Typical Representative culture collection center, 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 mTIGIT-hFc immune rat, get splenocyte and the myelomatosis IR983F cytogamy of the rear rat of immunity, screening hybridoma, the high affine mTIGIT-hFc of monoclonal anti physical efficiency specificity of its secretion, the cell strain called after mTIGIT-mAb-13G6 that screening is obtained, deposit number is CCTCC NO:C201299.
The anti-mouse TIGIT monoclonal anti physical efficiency of being secreted by cell strain mTIGIT-mAb-13G6 is efficiently blocked the combination of mTIGIT and mPVR.
In addition, anti-mouse TIGIT monoclonal antibody of the present invention is also referred to as 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 hybridoma cell strain described in intraperitoneal inoculation.
Another aspect of the present invention relates to the blocking effect of monoclonal antibody to mTIGIT and mPVR combination.By monoclonal antibody of the present invention, detect blocking-up efficiency, result demonstration, monoclonal anti physical efficiency is blocked the combination of mTIGIT and mPVR efficiently.
Another aspect of the present invention relates to monoclonal antibody and detects in mTIGIT and apply at Western blot.With the expression of Identification of Monoclonal Antibodies mTIGIT albumen of the present invention, Western blot tests demonstration: monoclonal anti physical efficiency in Western blot level for detection of mTIGIT-hFc fusion rotein.
Another aspect of the present invention relates to monoclonal antibody in the application of ELISA level detection mTIGIT.By monoclonal antibody of the present invention, detect the mTIGIT-hFc of different concns, result shows: monoclonal anti physical efficiency detects mTIGIT-hFc albumen for ELISA.
Another aspect of the present invention relates to monoclonal anti physical efficiency and detects mTIGIT for Flow Cytometry.Can be 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 detects the expression of mTIGIT for Flow Cytometry.
The monoclonal antibody that the invention still further relates to anti-mouse TIGIT of the present invention is in the application for the preparation of detecting in the test kit of 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, the monoclonal antibody that described test kit comprises anti-mouse TIGIT of the present invention.
The invention still further relates to the method for mTIGIT of detection a kind of, described method comprises the steps: that (1) hatch monoclonal antibody of the present invention with separated cell 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 testing sample, and described positive reaction can judge by the immune marking, ELISA or Flow cytometry.
In the present invention, term " specificity of monoclonal antibody " refers to defined epitope on monoclonal antibody identification antigen or antigenic determinant 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 mTIGIT-hFc fusion rotein, according to method described herein, being prepared with specific monoclonal antibody 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 studying the function of 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
Accompanying drawing explanation
In 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) 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, the specific result of indirect elisa method checking monoclonal antibody to 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, 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 detects the result of mTIGIT for Western blot.
Fig. 7, monoclonal antibody of the present invention detects the result of mTIGIT for ELISA.
Fig. 8, monoclonal antibody of the present invention detects the result of mTIGIT for FACS.
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.
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.
Sequence table explanation
Embodiment
Following examples will contribute to those of ordinary skill in the art further to understand the present invention, but not limit in any form the present invention.
Experimental technique in embodiment, if no special instructions, all adopts this area routine techniques, and experiment reagent is commercially available prod.
Embodiment 1: the structure of carrier for expression of eukaryon pcDNA3-mTIGIT-hFc
From pMD18-T-total length mouse TIGIT carrier, (pMD18-T is purchased from Takara, Code:D103A) pcr amplification mouse TIGIT extracellular fragment encoding sequence in, by sequence fragment and pcDNA3-hFc recombinant plasmid, (pcDNA3 is purchased from Invitrogen, numbering V790-20) after cutting with Xho I place enzyme, EcoRI is connected, connect product and transform DH5 α intestinal bacteria, coated plate grow overnight, and the bacterial strain that screening is obtained carries out PCR detection, PCR is identified to 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)
By pcDNA3-mTIGIT-hFc carrier transfection 293A cell (purchased from Shanghai cell bank, catalog number (Cat.No.): GNHu 1), collect serum-free culture thing supernatant, 30kD film (purchased from the green skies) concentrates supernatant, with Protein G purification column (purchased from GE Healthcare, HiTrap Protein G HP, article No.: 1700404-01) concentrated supernatant is carried out to the enrichment of target protein, then use acid PBS (pH2.8) to carry out wash-out, again the protein solution of wash-out is neutralized with alkaline Tris damping fluid (pH9.0), obtain mTIGIT-hFc protein solution.Result as shown in Figure 2 A.
2, the evaluation of fusion rotein
The mTIGIT-hFc albumen of purifying is carried out to SDS-PAGE & Western Blot to be identified; Result is 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, purchased from Sigma, article No. F5881-10ML) mix, the final concentration of mTIGIT-hFc is 100 μ g/ml, and mixture is fully mixed to formation water-in-oil, gets rat (purchased from Shanghai Si Laike, male, 8 week age) 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 immunity in 2 weeks is once.After immunity 5 times, the serum titer that enzyme-linked immunosorbent assay (ELISA) detects rat is very high, after rat is put to death, gets Rats Spleen, crosses 200 order cell sieves, collects the splenocyte filtering, and after standing several minutes, draws 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, with PBST (0.05%Tween20-PBS pH 7.4), clean 3 times, 1%BSA sealing, hatches 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 hatch 1 hour for 37 ℃ in 100 μ l/ holes.PBST cleans 3 times, and every hole adds the sheep anti-mouse antibody (purchased from Boster company, article No. BA1050) of horseradish peroxidase (HRP) mark of 100 μ l dilution in 1: 10000, hatches 1 hour for 37 ℃.After cleaning, add tmb substrate liquid (purchased from eBioscience company, article No. 00-4201-56), 100 μ l/ holes, lucifuge colour developing 15 minutes, adds stop buffer (2MH
2sO
4) 50 μ l/ holes, after termination, detect immediately OD490.
2, the preparation of feeder cell
In cytogamy the day before yesterday, prepare rat peritoneal macrophages, as feeder layer cells.Concrete grammar is as follows: normal rat dislocation in 8 week age is put to death, and 75% alcohol disinfecting, exposes belly, cuts off peritonaeum.With 10ml syringe, draw the incomplete nutrient solution of 5ml DMEM (purchased from Gibco company) 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 96 Zhong，Mei hole, hole 100 μ l, puts into cell culture incubator, and 37 ℃, 5%CO
2under condition, cultivate.
3, the preparation of cytogamy and hybridoma
Get splenocyte (10-15 * 10 that prepare
7) and myeloma cell IR983F (purchased from logical (Shanghai) bio tech ltd, 5 * 10 of sending
7) in serum-free DMEM substratum, fully mix, 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), after 60 seconds, add serum-free DMEM substratum (purchased from Gibco company), the centrifugal cell of collecting precipitation afterwards, adds HAT to select substratum resuspended.HAT selects the DMEM substratum of substratum for comprising 20%FCS (calf serum), 10mM sodiumhypoxanthine (xanthoglobulin), 40mM aminopterin (aminopterin), 1.6mMthymidine (thymus pyrimidine).
(the cell suspension merging is added to previously prepared feeder cell, the feeder cell of preparation in above-mentioned 2) in, fused cell is after HAT selection culture medium culturing one week, 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 object antibody and carries out ELISA detection.With the DMEM nutrient solution that contains 10% calf serum, replace HT nutrient solution cultivates simultaneously.
Adopt limiting dilution method to carry out subcloning the hybridoma of the generation monoclonal antibody of the present invention obtaining, after many screenings of ELISA, obtain hybridoma cell strain mTIGIT-mAb-13G6, 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 is preserved in (address: Wuhan, China, Wuhan University, postcode: 430072), deposit number is CCTCC NO:C201299, Chinese Typical Representative culture collection center on September 25th, 2012.
3, the preparation of monoclonal antibody
During 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 supernatant.They are two years old, described hybridoma can be expelled to the nude mice abdominal cavity with paraffin preimmunization, by extracting nude mice ascites, extract acquisition monoclonal antibody, 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, produces ascites for about 7-10 days, collects nude mice ascites, and centrifugal receipts supernatant is ascites antibody.The antibody obtaining by aforesaid method, can purify by Protein G affinity chromatography method, and the purity of antibody is identified with SDS-PAGE.As shown in Figure 3, the purity of purified monoclonal antibody approaches 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 with PBST (0.05%Tween20-PBS, pH 7.4), clean 3 times, and 1%BSA sealing, hatches 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, purchased from Chinese Academy of Sciences's Shanghai cell bank) culture supernatant or ascites works as negative control, establishes PBS for zeroing hole, 100 μ l/ holes, hatch 1 hour for 37 ℃.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 purchased from Absea) of 100 μ l horseradish peroxidase (HRP) marks, hatches 1 hour for 37 ℃.After cleaning, add tmb substrate liquid (purchased from eBioscience company, article No. 00-4201-56), 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 PBS hole, when test antibody detected value/negative control value (ratio) is greater than 2.1, be made as the positive, take the titre that the positive hole of greatest dilution is antibody.
2, BIAcore 3000 measures the affinity costant of monoclonal antibody.
Put into CM5 chip, Dock, makes moving phase with PBS, twice of sensitization.First inject 50mMNaOH+0.05%SDS (10 μ l/min, 2min), then inject 50mM NaOH (10 μ l/min, 2min) and rinse chip surface.First grope suitable mTIGIT-hFc concentration and pH, 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 mixes by 1: 1 volume ratio) (5 μ l/min, 7min,) activation chip, with the NaAc of PH 5.0 by antigen (, mTIGIT-hFc) be diluted to 10 μ g/ml, the antigen of injection dilution, starts to carry out antigenic mark, passage of mark, stays another passage to do blank.With thanomin sealing, off-period is identical when flow velocity is with activation.1 * PBS sensitization twice by different antibodies concentration determination mark effect, is groped regeneration condition simultaneously, can determine that 50mM NaOH is proper regeneration condition.By 13G6 monoclonal antibody (, 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 of μ g/ml concentration, setting program is measured the avidity of 13G6 monoclonal antibody to antigen mTIGIT-hFc.Obtain carrying out matching after 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 |
| 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: build recombinant vectors pcDNA3-mTIGIT, pcDNA3-hTIGIT, pcDNA3-mCD226 and pcDNA3-mCD96, by the recombinant vectors difference transfection 293T cell (293T cell is purchased from Shanghai cell bank) building), the mouse IgG2a of the Zai Yong PE-Chinese People's Anti-Japanese Military and Political College is (purchased from eBioscience, article No. 12-4817) be two anti-detection 13G6 monoclonal antibodies, result as shown in Figure 4 A, 13G6 only can specific recognition mTIGIT molecule, to its excess-three kind molecule without identification, illustrate that 13G6mAb is the monoclonal antibody specific for mTIGIT.
By 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) (purchased from Absea) of HRP mark resists and carries out ELISA detection as two, and 13G6mAb is not substantially in conjunction with eGFP-hFc, as shown in Figure 4 B.
Adopt the method for immunoblotting to carry out specific detection, we using this monoclonal antibody as primary antibodie, the mouse IgG of the Chinese People's Anti-Japanese Military and Political College (OLIGOAB02H) (purchased from Absea) of HRP mark resists and carries out Western blot detection as two, detect fusion rotein mTIGIT-hFc and eGFP-hFc, through Western blot, identify, as shown in Figure 4 C, at molecular weight 41kDa (mTIGIT-hFc), located a specific band, and located there is no specific band in 55kDa left and right (eGFP-hFc).Further proving that 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
13G6mAb cell culture supernatant is first hatched with 1 μ g mTIGIT-hFc albumen, and 4 ℃, 20min; Use again the resuspended 293T-mPVR cell of mixtures incubated (293T cell is purchased from 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 (purchased from BD Bioscience), 4 ℃, 20min, proceeds to streaming pipe, detects (BD FACSCalibur) detect with small-sized flow cytometer.As shown in Figure 5 B, contrast is complete to be blocked and not blocking-up group result, and 13G6mAb can block the combination of mTIGIT and mPVR substantially completely, illustrates that this monoclonal antibody has efficient blocking ability.Embodiment 5: the application of anti-mouse TIGIT monoclonal antibody
1) for Western blot, detect mTIGIT
By 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 (purchased from BOSTER, article No.: BA1058) resist and carry out Western blot detection as two, as Fig. 6, this monoclonal antibody can detect the mTIGIT-hFc albumen in the full lysate of 293A-mTIGIT-hFc cell, in 293A-eGFP-hFc total protein, signal do not detected.
2) for ELISA, detect mTIGIT
With the fusion rotein mTIGIT-hFc of different concns, be coated with 96 orifice plates respectively, prepared monoclonal antibody is as primary antibodie, the mouse IgG of the Chinese People's Anti-Japanese Military and Political College (OLIGOAB02H) (purchased from Absea) of HRP mark is anti-as two, finally add TMB nitrite ion (purchased from eBioscience, article No. 00-4201-56), detect 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 ELISA requirement.The results are shown in as Fig. 7, show that this monoclonal antibody can be used in ELISA and detects mTIGIT albumen.
3) for Flow Cytometry, detect mTIGIT
FACS detects mTIGIT at clone 293A-mTIGIT (pcNDA3-mTIGIT plasmid transfection 293A cell, 293A is purchased from Shanghai cell bank) and the expression on 293A surface, prepared monoclonal antibody is as primary antibodie, the mouse IgG2a of the YongPE-Chinese People's Anti-Japanese Military and Political College (purchased from BD Bioscience) is anti-as two, positive cell (293A-mTIGIT) and negative cells (293A) with monoclonal antibody 13G6 marker expression mTIGIT of the present invention, as shown in Fig. 8 (A), prepared monoclonal antibody can be incorporated into 293A-mTIGIT cell surface, but not with 293A Cell binding.Contrast commercial Alexa 647/mTIGIT mark mTIGIT, as Fig. 8 (B), both ratios approach, and result shows that monoclonal antibody of the present invention can be 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 described, but will be understood by those skilled in the art that, under the condition not deviating 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.
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