CN103936853B - A kind of detection TIM-3 test kit and using method thereof - Google Patents
A kind of detection TIM-3 test kit and using method thereof Download PDFInfo
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Abstract
The invention belongs to technical field of bioengineering, disclose a kind of detection TIM 3 test kit and using method, invention additionally discloses the monoclonal antibody for detecting Tim 3 or its fragment, described antibody or its fragment include light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and each CDR has aminoacid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.The monoclonal antibody that the present invention provides can combine with people Tim 3 by specificity, with the test kit of detection people Tim 3 prepared by described antibody, has higher sensitivity, it is possible to detect the people Tim 3 of low concentration.
Description
Technical field
The invention belongs to technical field of bioengineering, particularly to monoclonal antibody, its preparation method and the application in preparation detection TIM-3 reagent of anti-human Tim-3.
Background technology
Tim-3 structurally belongs to T cell immune globulin Pseudobulbus Bletillae (Rhizoma Bletillae) mucin (T cell Immunoglob μ Lin domain and Mucin domain protein, Tim) family member.Tim-3 is specific expressed in Th1, Th17 effector lymphocyte surface of activation, and is not expressed in Th2 cell.Know galactose-binding protein-9 (Galectin-9, Gal-9) it is the native ligand of Tim-3, this molecule wide expression is in periphery immune system, Tim-3 molecular specificity on Gal-9 with Th1, Th17 cell is combined, the tune that can cause the latter is died, lower immunoreation, inducing immune tolerance.Except the Tim-3 molecule of film combining form, whether the Tim-3 molecule of solubility, and how to affect process of immune regulation be the content that it be not immediately clear.At present, research confirms that Tim-3 molecule is mainly by the different CD4 of regulation+The function wide participation of the T cell subgroup immunne response processes such as autoimmune disease, transplant rejection, infection.
At present, research confirms that the exception that Tim-3 expresses has close relationship with numerous disease, as research finds that in the patient of HIV, the patient of HCV infection, the patient of HBV infection, pyemic patient and some tumor patient T cell, the expression of Tim-3 raises.Such as, compared with normal mouse, in sepsis mouse model abdominal cavity, on T cell, DC cell, Tim-3 expression significantly increases.Tim-3 can mediate T effector lymphocyte's tune and die, transmission negativity regulation signal, may result in the immunologic function paralysis of body.Research shows, the factor that any blocking-up Tim-3/Gal-9 combines all can make Th1, Th17 cell avoid death, strengthens the activity of T effector lymphocyte, recovers immunologic function.
On the one hand, in the diseases such as some autoimmune diseases such as systemic lupus erythematosus (sle) (SLE), asthma, the rising expressed due to Gal-9 or Tim-3, the function causing Th1 cell is suppressed, and then break internal immunologic balance, cause the increased activity of pathologic Th2 cell, cause the morbidity of disease.In this case, the recovery that the factor that any blocking-up Tim-3/Gal-9 combines all can be conducive to vivo immunization to balance, alleviate the process of disease.Inject anti-Tim-3 antibody such as to asthmatic model animal or restructuring Tim-3 fusion protein all can combine by blocking Tim-3/Gal-9, strengthen Th1 cytoactive, correct by symptoms of asthma cell-mediated for Th2, recover internal Th1/Th2 cell balance.On the other hand, in some autoimmune diseases such as inflammatory bowel and type i diabetes model, research finds the increased activity of the blocking-up Tim-3 function of internal Th1 effector lymphocyte, having increased the weight of autoimmune lesions further, this result demonstrates again that in the maintenance of Tim-3 immunologic balance in vivo and plays a significant role.
The studies above data shows, Tim-3 path has important immunoloregulation function, and the exception that Tim-3 expresses has substantial connection with generation, the development of multiple disease.Although research shows that the neutralizing antibody of Tim-3 has played good intervention effect in some disease model, but, the most not Tim-3 antibody of listing.Therefore it provides the Tim-3 antibody of a kind of high specificity, study of pathogenesis, medical diagnosis on disease and treatment to some disease have great importance.
Summary of the invention
In view of this, the invention provides monoclonal antibody, its preparation method and the application of a kind of anti-human Tim-3, monoclonal antibody or its fragment energy specificity of this anti-human Tim-3 are combined with people Tim-3, may be used for the test kit of preparation detection people Tim-3, gained test kit has higher sensitivity, it is possible to detect the people Tim-3 of low concentration.
In order to realize the goal of the invention of the present invention, the present invention adopts the following technical scheme that:
The invention provides monoclonal antibody or its fragment of a kind of anti-human Tim-3, it includes light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3；
Light chain CDR1 has aminoacid sequence shown in SEQ ID NO:1；
Light chain CDR2 has aminoacid sequence shown in SEQ ID NO:2；
Light chain CDR3 has aminoacid sequence shown in SEQ ID NO:3；
Heavy chain CDR1 has aminoacid sequence shown in SEQ ID NO:4；
Heavy chain CDR2 has aminoacid sequence shown in SEQ ID NO:5；
Heavy chain CDR3 has aminoacid sequence shown in SEQ ID NO:6.
Preferably, the monoclonal antibody of present invention offer or its fragment, its variable region of light chain includes light chain CDR1, light chain CDR2 and light chain CDR3, and its variable region of heavy chain includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3；
Light chain CDR1 has aminoacid sequence shown in SEQ ID NO:1；
Light chain CDR2 has aminoacid sequence shown in SEQ ID NO:2；
Light chain CDR3 has aminoacid sequence shown in SEQ ID NO:3；
Heavy chain CDR1 has aminoacid sequence shown in SEQ ID NO:4；
Heavy chain CDR2 has aminoacid sequence shown in SEQ ID NO:5；
Heavy chain CDR3 has aminoacid sequence shown in SEQ ID NO:6.
Preferably, the monoclonal antibody of present invention offer or its fragment, its variable region of light chain has aminoacid sequence shown in SEQ ID NO:7.
Preferably, the monoclonal antibody of present invention offer or its fragment, its variable region of heavy chain has aminoacid sequence shown in SEQ ID NO:8.
In some embodiments of the invention, monoclonal antibody or its fragment that the present invention provides are monoclonal antibody, and its variable region of light chain has aminoacid sequence shown in SEQ ID NO:7；Its variable region of heavy chain has aminoacid sequence shown in SEQ ID NO:8, named L3A monoclonal antibody.
Present invention also offers the preparation method of a kind of monoclonal antibody or its fragment, comprise the following steps:
Step 1: acquisition has the DNA molecular of nucleotide sequence shown in SEQ ID NO:9, has the DNA molecular of nucleotide sequence shown in SEQ ID NO:10；
Step 2: take step 1 gained and there is DNA molecular and the fusion of the first expression vector of nucleotide sequence shown in SEQ ID NO:9, build the first recombinant expression carrier；Take step 1 gained and there is DNA molecular and the fusion of the second expression vector of nucleotide sequence shown in SEQ ID NO:10, build the second recombinant expression carrier；Maybe by there is the DNA molecular of nucleotide sequence shown in SEQ ID NO:9 and there is DNA molecular and the fusion of the 3rd expression vector of nucleotide sequence shown in SEQ ID NO:10, build the 3rd recombinant expression carrier；
Step 3: taking step 2 gained the first recombinant expression carrier and step 2 gained the second recombinant expression carrier, cotransfection proceeds to host cell；Or step 2 gained the 3rd recombinant expression carrier is proceeded to host cell；Express, purification, to obtain final product；
The variable region of light chain of this monoclonal antibody or its fragment has aminoacid sequence shown in SEQ ID NO:7；Variable region of heavy chain has aminoacid sequence shown in SEQ ID NO:8.
In some embodiments of the invention, in the preparation method step 2 that the present invention provides, the first expression vector is the pcDNA3.1 plasmid containing antibody light chain constant region gene.
In the other embodiment of the present invention, in the preparation method step 2 that the present invention provides, the second expression vector is the pcDNA3.1 plasmid containing heavy chain constant region gene.
In the other embodiment of the present invention, in the preparation method step 2 that the present invention provides, the 3rd expression vector is the pcDNA3.1 plasmid containing antibody constant region gene.
In the other embodiment of the present invention, in the preparation method step 3 that the present invention provides, host cell is mammalian cell.
In some embodiments of the invention, the preparation method of monoclonal antibody L3A that the present invention provides comprises the following steps:
Step 1: obtain the DNA molecular of coding light chain, this DNA molecular comprises and has nucleotide sequence shown in SEQ ID NO:9；Obtaining the DNA molecular of encoding heavy chain, this DNA molecular comprises and has nucleotide sequence shown in SEQ ID NO:10；
Step 2: take the DNA molecular of step 1 gained coding light chain, merge with the first expression vector, build the first recombinant expression carrier；The DNA molecular and the second expression vector that take step 1 gained encoding heavy chain merge, and build the second recombinant expression carrier；
Step 3: taking gained step 2 gained the first recombinant expression carrier, the second recombinant expression carrier, cotransfection proceeds in mammalian cell, expresses, purification, to obtain final product.
Present invention also offers acquisition monoclonal antibody L3A variable region of light chain, weight chain variable region nucleotide sequence and the method for aminoacid sequence, particularly as follows:
1, the structure of anti-human Tim-3 monoclonal antibody hybridoma cell strain
First, it is thus achieved that people's Tim-3 albumen, immunity BALB/c mouse, carry out cell fusion by conventional method.Positive cell clone sub-clone the most repeatedly is screened, until all Hybridoma Cell Culture supernatants are detected as 100% positive with indirect elisa method.Gained hybridoma has carried out chromosome karyotype analysis, and hybridoma chromosome average number is 108, proves that the immunoglobulin hypotype secreted by gained hybridoma is IgG2a with the experiment of two-phase agar diffusion.
2, the variable region of light chain of monoclonal antibody L3A, the fishing of heavy chain variable region gene take and variable region of light chain, the determination of heavy chain variable region gene
Extract the RNA of gained hybridoma, through RT-PCR, fish chain variable region gene and the heavy chain variable region gene taking antibody respectively with specific primer.Use conventional method that gained chain variable region gene proceeds to carrier, transformed competence colibacillus antibacterial, the single bacterium colony of picking after cultivation, after extracting plasmid PCR qualification correctly, carry out DNA sequencing；Derived heavy chain variable region gene is proceeded to carrier, transformed competence colibacillus cell, the single bacterium colony of picking after cultivation, after extracting plasmid PCR qualification correctly, carry out DNA sequencing；Through sequence analysis, comparison, it is thus achieved that the chain variable region gene of monoclonal antibody L3A and heavy chain variable region gene.The chain variable region gene sequence of gained monoclonal antibody L3A is nucleotide sequence shown in SEQ ID NO:9, and heavy chain variable region gene sequence is nucleotide sequence shown in SEQ ID NO:10.
3, monoclonal antibody L3A light chain, the determination of heavy chain variable amino acid sequence
The light chain of encoding human Tim-3 monoclonal antibody L3A, weight chain variable region nucleotide sequence are translated as the aminoacid sequence of its coding with the online software of www.expasy.org, and the light chain of monoclonal antibody L3A, heavy chain variable amino acid sequence are respectively shown in aminoacid sequence shown in SEQ ID NO:7, SEQ ID NO:8 shown in aminoacid sequence.Determine that the aminoacid sequence of complementary determining region light chain CDR1, light chain CDR2 and light chain CDR3 in monoclonal antibody L3A light-chain variable sequence is respectively as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in sequence table according to Kabat data base.The aminoacid sequence of complementary determining region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 in weight chain variabl area sequence is respectively as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in sequence table.
Present invention also offers monoclonal antibody or the DNA molecular of its fragment that the invention of a kind of code book provides, it has nucleotide sequence shown in SEQ ID NO:9, and the variable region of light chain of this monoclonal antibody or its fragment has aminoacid sequence shown in SEQ ID NO:7.
Present invention also offers monoclonal antibody or the DNA molecular of its fragment that the invention of a kind of code book provides, it has nucleotide sequence shown in SEQ ID NO:10, and the variable region of heavy chain of this monoclonal antibody or its fragment has aminoacid sequence shown in SEQ ID NO:8.
The present invention has successfully cloned from the people's Tim-3 monoclonal antibody hybridoma cell cultivated that antibody is light, heavy chain variable region gene.Gained chain variable region gene and the correct mouse antibody variable region of heavy chain variable region gene codified.People's Tim-3 monoclonal antibody L3A chain variable region gene of being cloned into based on above-mentioned, heavy chain variable region gene, can build and express multiple little molecular gene engineered antibody, such as single-chain antibody, single domain antibody, chimeric antibody, Fab antibody, antibody fusion protein etc.；Based on the polypeptide coded by said gene or protein, can cross-link multiple bioactive molecule, preparation is for the immune detection instrument of people's Tim-3 expression detection, preparation diagnosis and the medicine for the treatment of the caused disease of Tim-3 high expressed.
Present invention also offers a kind of monoclonal antibody or its fragment and preparing the application in the immune detection instrument detecting people Tim-3, this monoclonal antibody or its fragment include light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3；
Light chain CDR1 has aminoacid sequence shown in SEQ ID NO:1；
Light chain CDR2 has aminoacid sequence shown in SEQ ID NO:2；
Light chain CDR3 has aminoacid sequence shown in SEQ ID NO:3；
Heavy chain CDR1 has aminoacid sequence shown in SEQ ID NO:4；
Heavy chain CDR2 has aminoacid sequence shown in SEQ ID NO:5；
Heavy chain CDR3 has aminoacid sequence shown in SEQ ID NO:6.
Preferably, the immune detection instrument in the application that the present invention provides is test kit, chip or reagent paper.
In some embodiments of the invention, the immune detection instrument in the application that the present invention provides is test kit.
A kind of monoclonal antibody or the test kit of its fragment, this monoclonal antibody or its fragment provided containing the present invention is provided and includes light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3；
Light chain CDR1 has aminoacid sequence shown in SEQ ID NO:1；
Light chain CDR2 has aminoacid sequence shown in SEQ ID NO:2；
Light chain CDR3 has aminoacid sequence shown in SEQ ID NO:3；
Heavy chain CDR1 has aminoacid sequence shown in SEQ ID NO:4；
Heavy chain CDR2 has aminoacid sequence shown in SEQ ID NO:5；
Heavy chain CDR3 has aminoacid sequence shown in SEQ ID NO:6.
In some embodiments of the invention, the test kit that the present invention provides includes L3A monoclonal antibody, rabbit against human T im-3 polyclonal antibody and HRP labelling goat anti-rabbit antibody.
The monoclonal antibody L3A high specificity that the present invention provides, it is possible to specificity combines with people Tim-3, when it is applied to the test kit of preparation detection people Tim-3, significantly improves the sensitivity of test kit.Test result indicate that, the test kit that the present invention provides can detect the content of Tim-3 in biological sample delicately, and its sensitivity reaches 0.1ng/mL～1000ng/mL.
In some embodiments of the invention, the preparation method of the rabbit against human T im-3 polyclonal antibody in the test kit that the present invention provides includes:
Obtain people's Tim-3 antigen；
After income earner Tim-3 immunity rabbit, gather antiserum, purification, to obtain final product.
Present invention also offers the using method of mentioned reagent box, comprise the following steps:
Under the conditions of step 1:4 DEG C, it is coated L3A monoclonal antibody 16h～18h；
Step 2: add biological sample, hatch 1h for 37 DEG C；
Step 3: add rabbit against human T im-3 polyclonal antibody, hatch 1h for 37 DEG C；
Step 4: add HRP labelling goat anti-rabbit antibody, after 37 DEG C hatch 0.5h, colour developing, terminate, detection；
Step 5: judge the concentration of people Tim-3 contained in biological sample according to testing result.
The invention provides monoclonal antibody or its fragment of a kind of anti-human Tim-3.The monoclonal antibody of this anti-human Tim-3 or its fragment include light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and wherein light chain CDR1 has aminoacid sequence shown in SEQ ID NO:1；Light chain CDR2 has aminoacid sequence shown in SEQ ID NO:2；Light chain CDR3 has aminoacid sequence shown in SEQ ID NO:3；Heavy chain CDR1 has aminoacid sequence shown in SEQ ID NO:4；Heavy chain CDR2 has aminoacid sequence shown in SEQ ID NO:5；Heavy chain CDR3 has aminoacid sequence shown in SEQ ID NO:6.Test result indicate that, monoclonal antibody or its fragment that the present invention provides can combine with people Tim-3 by specificity, may be used for the test kit of preparation detection people Tim-3, and gained test kit has higher sensitivity, it is possible to detect the people Tim-3 of low concentration.
Accompanying drawing explanation
Fig. 1 shows the result schematic diagram of the antiserum titre of the polyclonal antibody of the rabbit against human T im-3 using the indirect ELISA method detection embodiment of the present invention 5 to prepare, and wherein curve 1 shows monoclonal antibody and people's Tim-3 binding curve of the prepared rabbit against human T im-3 of embodiment 5；Curve 2 shows matched group serum and people's Tim-3 binding curve；
Fig. 2 shows the sensitivity technique result of the prepared test kit of the embodiment of the present invention 6, and wherein, curve 1 shows that experimental group testing result, curve 2 show matched group testing result；
Fig. 3 shows the result of people's Tim-3 level in the different biological specimen of test kit detection using embodiment 6 to prepare in the embodiment of the present invention 8, wherein Fig. 3 A is adult samples: sample 1: normal healthy controls, sample 2:TID (type 1 diabetes), sample 3:SLE (systemic lupus erythematosus (sle))；Fig. 3 B is child: sample 4: normal healthy controls, sample 5: asthma.
Fig. 4 shows the result of people's Tim-3 level in the test kit detection using embodiment 6 to prepare in the embodiment of the present invention 9 pyemia patients serum in various degree.
Detailed description of the invention
The invention discloses monoclonal antibody, its preparation method and the application of a kind of anti-human Tim-3.Those skilled in the art are referred to present disclosure, implement this preparation method, it is thus achieved that the monoclonal antibody of anti-human Tim-3, it is accordingly required in particular to be pointed out that, all similar replacements and change apparent to those skilled in the art, they are considered as being included in the present invention.The preparation method of the present invention is described by preferred embodiment, and this paper preparation method substantially can be modified in without departing from present invention, spirit and scope or suitably change and combine by related personnel, realizes and applies the technology of the present invention.
Reagent used in the monoclonal antibody of anti-human Tim-3, its preparation method and application that the present invention provides and raw material all can be buied by market.
In order to make those skilled in the art better understood when technical scheme, below in conjunction with embodiment, the present invention it is expanded on further:
The structure of embodiment 1 anti-human Tim-3 monoclonal antibody hybridoma cell strain
1. material
Freund's complete adjuvant and freund 's incomplete adjuvant, colour reagent TMB:Sigma Products；20% hyclone: Heng Shengma biotechnology research institute of Beijing unit product；Serum-free RPMI1640:Gibco Products；SP2/0 cell: ATCC introduces, and Institute of Basic Medical Sciences, A Cademy of Military Medical Sciences, preserves；BALB/c and C57BL/6 mice: Military Medical Science Institute's Experimental Animal Center provides；Recombined human Tim-3 (rhTim-3) is prepared by Institute of Basic Medical Sciences, A Cademy of Military Medical Sciences,.Remaining reagent is commercial.
2. method and result
(1) BALB/c mouse immunity.Select the female BAl BIc/c mice 6 of 4～6 week old, with 100 μ g ricins (Sigma company) in groin subcutaneous inoculation, the first pin Freund Freund's complete adjuvant, the second pin Freund Freund's incomplete adjuvant, immunity in every 3 weeks 1 time, altogether immunity 3 times.Tail vein blood after 3rd immunity, detects antibody production with indirect ELISA, in fusion first 3 days, with 100 μ g ricin abdominal cavity booster immunizations once, within the 3rd day, merges.
(2) cell merges.The mice of immunity is plucked de-neck after eyeball put to death, aseptic win mouse boosting cell, carry out cell fusion according to a conventional method.Method particularly includes: after 1. the mice of immunity being plucked eyeball blood-letting, de-neck is put to death, 75% alcohol-pickled 3min, and aseptic taking-up spleen grinds individual cells suspension with 200 mesh steel meshes, and serum-free RPMI1640 washes twice and counts；2. collect the SP2/0 cell of exponential phase, wash twice with serum-free RPMI1640 and count；3. in SP2/0 cell: splenocyte=1: ratio two kinds of cells of mixing of 5, wash 1 time with RPMI1640, abandon most supernatant, gently cell is broken up；4. in the 1min time, it is slowly added to 1mL50%PEG (MWIt is 1500) solution, put 37 DEG C of water-bath 1min；5. serum-free RPMI16401mL, 5mL, 10mL are added respectively when 1min, 2min, 5min time；6. 800r/min is centrifuged 7min, abandons supernatant, is the most lightly hanged by cell；7. adding HAT (the Sigma)-RPMI1640 culture fluid containing 20%FCS, adjusting cell concentration is 2 × 106/ mL, drips after mixing and is being covered with trophocyte (1 × 104Cells/well) in 96 well culture plates (Gibco), 100 μ L/ holes, it is placed in the 5%CO of 37 DEG C2Incubator is cultivated.
(3) anti-human Tim-3 monoclonal antibody hybridoma cell is screened with indirect ELISA.It is coated elisa plate with 10 μ g/mL recombined human Tim-3 (rhTim-3), overnight and closes in 4 DEG C.It is sequentially added into cell culture supernatant to be measured (37 DEG C of 1h, PBST wash plate 4 times), and the 50 μ LHRP-GAM (37 DEG C of 45min, PBST wash plate 4 times) of 1: 500 dilution.After developing the color with tmb substrate, measure OD value in 450nm wavelength.
(4) hybridoma cell clone.With the positive cell clone sub-clone the most repeatedly of indirect elisa method screening, until all Hybridoma Cell Culture supernatants are detected as 100% positive.The cloning limiting dilution assay of hybridoma: 1. prepared trophocyte the same day or first 1 day in cloning: de-neck puts to death kunming mice, 75% alcohol-pickled sterilization skin, aseptic stripping skin of abdomen, syringe extraction 5mL1640 culture fluid injects mouse peritoneal, sucking-off abdominal cavity washing liquid after repeatedly rinsing, 96 orifice plates, every hole about 0.1mL is instilled after diluting with the RPMI-1640 adding 20% hyclone.2. the hybridoma taking cloning to be made moves in another sterile test tube, and accurate counting.3. limiting dilution assay carries out sub-clone.4. culture plate is placed in the 5%CO of 37 DEG C2Incubator is cultivated, after 5 days, examines under a microscope cell clone.Change liquid in good time, detection, take positive monoclonal cell strain and be enlarged cultivating, timely freeze-stored cell strain.
(5) determination of hybridoma immunoglobulin hypotype.With sheep anti-mouse igg 1, IgG2a, IgG2b and IgG3, culture supernatant after concentrating gained hybridoma makees the experiment of two-phase agar diffusion, result shows that above-mentioned Hybridoma Cell Culture supernatant can only be formed with sheep anti-mouse igg 2a antibody and combines band, it was demonstrated that the immunoglobulin hypotype secreted by the present embodiment gained hybridoma is IgG2a.By above-mentioned steps, screen people's Tim-3 monoclonal antibody, named L3A monoclonal antibody.
Embodiment 2 people's Tim-3 monoclonal antibody L3A variable region of light chain, the fishing of heavy chain variable region gene take
1. material
(1) variable region of light chain upstream and downstream primer
Light chain forward primer MuLC5, MuLC6 and MuLC7, its sequence is shown in sequence table SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 respectively；Light chain downstream primer MuCK, its sequence is shown in sequence table SEQ ID NO:14, and the primer concentration when PCR expands is 10pmol/ μ L.
(2) variable region of heavy chain upstream and downstream primer
Heavy chain forward primer MuHC5, MuHC6, MuHC7 and MuHC8, its sequence is shown in sequence table SEQ ID NO:15, SEQ ID NO16, SEQ ID NO:17 and SEQ ID NO:18；Heavy chain downstream primer MuIgG2a is shown in that SEQ ID NO:19 in sequence table, the primer concentration when PCR expands are 10pmol/ μ L.
(3) DNA fragmentation purification kit: OMEGA biotechnology Products；T4DNA ligase: New England Biolabs product；Carrier PGEM Teasy:Promega Products；Competence antibacterial JM109: purchased from Promega company.Remaining reagent source is with embodiment 1.
2. methods and results
(1) take the logarithm the cell strain 5 × (10 of monoclonal antibody L3A of embodiment 1 system of trophophase6～107) individual, centrifugal segregation supernatant, cell is uniformly upspring.Add 1mL TRIzol (Invitrogen) repeatedly to blow and beat and make cell fully crack, after vibrating 5 minutes, add 0.2mL chloroform, vibrate 15 seconds, room temperature placement 2～3min, 2 DEG C～8 DEG C of 12000r/min, centrifugal 15 minutes, taking supernatant in another new pipe, after adding 500 μ L isopropanols mixings, room temperature is placed 10 minutes, and 2 DEG C～8 DEG C of 12000r/min are centrifuged 10min.75% washing with alcohol precipitation, after drying, precipitates without the deionized water dissolving of RNase with 20 μ L.
(2) solution containing 1 μ g total serum IgE is taken, it is sequentially added into AMV5 × buffer 4 μ L, Oligo (dT) (500ng/ μ L) 0.5 μ L, 2.5mmol/L dNTP2 μ L, RNasin (50U/ μ L) 0.5 μ L, benefit deionized water is to 20 μ L, reverse transcription 2～5U, and 42 DEG C extend 1 hour.95 DEG C of degeneration 5min, put in ice bath, and products therefrom is cDNA the first chain.Respectively with primer amplified chain variable region gene and heavy chain variable region gene.
Chain variable region gene amplification system: take above-mentioned products therefrom, i.e. reverse transcription product 2 μ L, Taq enzyme 10 × buffer2 μ L, the each 1 μ L of light chain forward primer MuLC5, MuLC6 and MuLC7, downstream primer MuCK3 μ L, 2.5mmol/L dNTP4 μ L, adds Taq enzyme 1～2U, benefit deionized water to 50 μ L.PCR amplification condition is: 95 DEG C of degeneration 2 minutes, and loop parameter is: 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, totally 30 circulations, extends 10min, gained amplified production is labeled as amplified production 1 after 72 DEG C.
Heavy chain variable region gene amplification system: take above-mentioned products therefrom, i.e. reverse transcription product 2 μ L, Taq enzyme 10 × buffer2 μ L, the each 1 μ L of heavy chain forward primer MuHC5, MuHC6, MuHC7 and MuHC8, downstream primer MuIgG2a3 μ L, 2.5mmol/L dNTP4 μ L, adds Taq enzyme 1～2U, benefit deionized water to 50 μ L.PCR amplification condition is: 95 DEG C of degeneration 2 minutes, and loop parameter is: 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, totally 30 circulations, extends 10min, gained amplified production is labeled as amplified production 2 after 72 DEG C.
(3) DNA fragmentation to be reclaimed it respectively is isolated by amplified production 1 and amplified production 2 with agarose gel electrophoresis, i.e. target DNA fragment, the blob of viscose containing target DNA fragment is cut respectively under long wave ultraviolet light, put in centrifuge tube, labelling one by one, adding the change glue that three times of colloids are long-pending, 55 DEG C of water-baths are completely dissolved blob of viscose.It is dissolved in aqueous solution in the purpose fragment being separately recovered in amplified production 1 in target DNA fragment and amplified production 2 with DNA fragmentation purification kit and by the DNA fragmentation of purification, labelling respectively, by reclaim PCR primer in T4DNA ligase buffer by 2: 1 ratio (mol ratio) and carrier PGEM Teasy mix after, the T4DNA ligase adding 0.5U connects overnight in 16 DEG C, the cumulative volume of coupled reaction is 10 μ L, and one_to_one corresponding labelling.
(4) take above-mentioned gained and connect liquid 10 μ L, it is added in 200 μ L competence antibacterial JM109 and softly mixes, ice bath 30min, 42 DEG C of water-bath heat shocks 90 seconds, proceed to rapidly ice bath 2min, add 800 μ LLB culture medium, proceed to 37 DEG C of constant-temperature tables, 45min is shaken with the speed of 150r/min, 4000r/min is centrifuged 1min, discards 800 μ L of supernatant, takes precipitation and coats the solid LB flat board containing Amp (final concentration of 100 μ g/mL), flat-plate inverted is placed in 37 DEG C of incubators 12～18h, and one_to_one corresponding labelling.
(5) the single clone of picking in the above-mentioned flat board of gained, is inoculated in the LB culture medium containing penbritin (100 μ g/mL), and one_to_one corresponding labelling.37 DEG C of constant-temperature table 170r/min, shake overnight incubation.Taking 3mL bacterium solution respectively to add in 1.5mL Eppendorf pipe, 10000r/min is centrifuged 1min, abandons supernatant.Use plasmid extraction kit, precipitation thalline is resuspended in 100 μ L solution I, adds freshly prepared solution II 200 μ L, turn upside down for several times light and slowly, to liquid change limpid.Subsequently, add 150 μ L solution III, gently turn upside down and make liquid blending for several times, a large amount of white flock precipitate now occurs.4 DEG C, 12000r/min is centrifuged 5min, takes supernatant and adds to, in another Eppendorf pipe, add the saturated phenol of isopyknic Tris-HCl, and acutely after concussion, 12000r/min is centrifuged 5min, is moved to mutually by upper water in a new pipe.Add 500 μ L chloroforms, again extract once.Thereafter, careful absorption upper strata aqueous phase, move in a new pipe, add the dehydrated alcohol mixing of 2 times of volumes, place 3h in-20 DEG C.4 DEG C, 12000r/min is centrifuged 10min, abandons supernatant, washes precipitation 2 times with 70% ethanol, and drying at room temperature 20min is dissolved with 40 μ L aseptic double-distilled waters, carried out PCR qualification and DNA sequencing analysis.
The carrier of monoclonal antibody L3A chain variable region gene containing people Tim-3, the carrier of heavy chain variable region gene is constructed by above-mentioned steps.Through sequence alignment, it is thus achieved that the nucleotide sequence of the variable region of light chain of coding L3A monoclonal antibody and the nucleotide sequence of the variable region of heavy chain of coding L3A monoclonal antibody.The nucleotides sequence of the variable region of light chain of gained coding L3A monoclonal antibody is classified as nucleotide sequence shown in SEQ ID NO:9；The nucleotides sequence of the variable region of heavy chain of gained coding L3A monoclonal antibody is classified as nucleotide sequence shown in SEQ ID NO:10, and the aminoacid sequence of its correspondence is respectively aminoacid sequence shown in aminoacid sequence shown in SEQ ID NO:7, SEQ ID NO:8.
The expression of embodiment 3 monoclonal antibody L3A and purification
1. material
Protein A Sepharose CL4B post albumen post: this yuan of Beijing Zhenyang Bioisystech Co., Ltd product；PcDNA3.1 plasmid: Invitrogen；The source of remaining reagent and material is with embodiment 1.
2. method and result
Monoclonal antibody L3A chain variable region gene embodiment 2 obtained, light chain constant region gene loads pcDNA3.1 plasmid, must contain the carrier of monoclonal antibody L3A light chain gene；Monoclonal antibody L3A heavy chain variable region gene embodiment 2 obtained, weight chain constant area gene loads pcDNA3.1 plasmid, it is thus achieved that containing the carrier of monoclonal antibody L3A heavy chain gene.Carrier containing monoclonal antibody L3A light chain gene, the carrier cotransfection of heavy chain gene proceed to, in mammalian cell, express.Collect and express supernatant, add 1mL pH8.0,0.1mol/L phosphate buffer and adjust pH to 9.0 with 1mol/L TRIS-HCL.Mouse ascites is added in the Protein A Sepharose CL4B albumen post balanced with pH8.0,0.1mol/L phosphate buffer, washes pillar with above-mentioned buffer, until effluent can't detect foreign protein.With the citrate buffer solution eluting of pH3.0, collect effluent, and neutralize, with the PBS 72h of pH7.2,0.01mol/L with 1mol/L pH8.5TRIS-HCL buffer immediately.OD260, OD280 are surveyed in sampling on ultraviolet spectrophotometer, calculate protein content, in-20 DEG C of preservations after lyophilizing.
The specific detection of embodiment 4 monoclonal antibody L3A
1. material
Sepsis patient serum is the treated acquisition of blood being collected in PLA General Hospital septic；Normal human serum is the treated acquisition of blood being collected in normal person；ELISA is coated liquid, PBST, TMB, H2SO4, TNF-a, sulfur oxygen cyclase protein be commercially available, the source of remaining reagent and material is with embodiment 1.
2. method and result
Indirect ELISA method is used to investigate the specificity of the L3A monoclonal antibody that embodiment 3 prepares.Experiment sets matched group 1, matched group 2, matched group 3, experimental group, positive controls, negative control group.Wherein matched group 1 is coated irrelevant antigen TNF-a1 μ g/mL, and matched group 2 is coated irrelevant antigen sulfur oxygen cyclase protein (TRX) 1 μ g/mL；Matched group 3 is coated BSA1 μ g/mL；Experimental group is coated septic serum；Positive controls is coated people's TIM-3 antigen (i.e. recombined human Tim-3) 1 μ g/mL；Negative control group is coated normal human serum.Often group sample dilutes with being coated liquid, and each sample is coated 2 holes in elisa plate, and every hole 100 μ L, 4 DEG C overnight.PBST washes 5 times, closes, 200 μ L confining liquids, 37 DEG C, closes 2 hours, and PBST washes 5 times.Adding L3A monoclonal antibody 50 μ L by the final concentration of 10 μ g/mL, 37 DEG C are reacted 1 hour, and PBST washes 5 times.Add 300 × dilution anti-mouse antibody (KPL company), 37 DEG C of reaction 45min.PBST washes 7 times, adds the TMB in 50 μ L/ holes, and after room temperature lucifuge hatches 10min, the sulphuric acid of the 1mol/L adding 50 μ L/ holes terminates reaction, OD value at microplate reader detection 450nm, calculates the meansigma methods often organizing holes numerical value.Monoclonal antibody L3A specific detection the results are shown in Table 1.
Table 1 monoclonal antibody L3A specific detection result
According to data in table 1, the OD450 value difference of four groups of negative control group, matched group 1, matched group 2 and matched group 3 (three unrelated protein comparisons) is not different notable, illustrates that the monoclonal antibody L3A not nothing to do with albumen that embodiment 3 prepares combines；Compared with negative control group, positive controls and experimental group OD450 value difference are different and notable, present obvious positive findings.Compared with positive control, experimental group is suitable with the OD450 value of positive controls, containing people's Tim-3 antigen in experimental group, illustrate that monoclonal antibody L3A that embodiment 3 prepares can combine with people Tim-3 by specificity, and not with other protein binding, may be used for preparation detection people Tim-3 immune detection instrument.
The preparation of embodiment 5 rabbit against human T im-3 polyclonal antibody
1. material
Grow up rabbit: male, body weight 2-3kg, and Military Medical Science Institute's Experimental Animal Center provides；Freund's complete adjuvant and freund 's incomplete adjuvant: Sigma Products；Remaining reagent and material source are with embodiment 1.
2. method and result
(1) rabbit immunity.Being mixed with adjuvant 1: 1 by people's Tim-3 antigen (PBS dissolving), emulsifying is complete.For the first time with the immunity of complete spoke adjuvant (CFA), second, third time full spoke adjuvant IFA that cannots be used up is immune, At intervals of two to three weeks.Selecting adult male rabbit 2, before immunity, every rabbit is through the ear vein each 1mL of blood sampling, separates serum, one by one labelling, for check sample.Immunization method is subcutaneous multi-point injection, accumulated dose 2mL (1mL people's Tim-3 antigen+1mL adjuvant)/and only.After final injection the 7th day, ear vein blood sampling 1mL, separate serum, one by one labelling, titrate titer by tube agglutination test, testing result is that both sero-fast titers are respectively 1: 3000 and 1: 4000 (more than 1: 2000 can use), all meets the requirements.
(3) antiserum collection.Take the rabbit that antiserum titre is 1: 4000, carotid artery blood-letting, and be collected in aseptic conical flask and solidify rear 4 DEG C of refrigerator overnight, separate upper serum.Then 2500r/min is centrifuged 10min, collects upper serum.The serum collected is identified after purification, and subpackage in a small amount ,-20 DEG C frozen.
(4) ELISA method detection antiserum titre.Experiment is divided into two groups, for experimental group and matched group；Experimental group and matched group are coated people Tim-3 antigen 1 μ g/mL, and every hole 100 μ L, 4 DEG C overnight.PBST washes 5 times, closes, 200 μ L confining liquids, 37 DEG C, closes 2 hours, and PBST washes 5 times.Being then respectively adding the antiserum of different dilution (3) gained and matched group serum to investigate the antiserum 50 μ L of the rabbit against human T im-3 that the present embodiment obtains, 37 DEG C are reacted 1 hour, and PBST washes 5 times.Add HRP labelling goat anti-rabbit antibodies, 37 DEG C of reaction 60min.PBST washes 7 times, adds the TMB in 50 μ L/ holes, and after room temperature lucifuge hatches 10min, the sulphuric acid of the 1mol/L adding 50 μ L/ holes terminates reaction, and microplate reader OD450nm detects.After deducting blank control wells absorption value, map with antiserum extension rate with OD450nm value, obtain rabbit against human T im-3 antiserum and the combination situation of people Tim-3 that the present embodiment prepares.With (sample light absorption value-blank absorbency)/(negative light absorption value-blank absorbency) ＞ 2.1, i.e. it is considered positive, calculates the rabbit against human T im-3 antiserum titre of the present embodiment gained.The rabbit against human T im-3 antiserum (i.e. polyclonal antibody) that the present embodiment prepares is combined situation with people's Tim-3 antigen and sees Fig. 1, contrast matched group, calculate according to above-mentioned formula, the sero-fast titer obtaining the rabbit against human T im-3 that the present embodiment obtains is 1: 3000, and i.e. the titer of the polyclonal antibody of the rabbit against human T im-3 that the present embodiment obtains is 1: 3000.
The preparation and application of embodiment 6 test kit
1. the preparation of test kit
The test kit that the present invention provides includes following components: L3A monoclonal antibody, rabbit against human T im-3 polyclonal antibody, horseradish peroxidase substrate buffer solution, protein standard substance recombined human Tim-3 (100ug/mL, 0.1mL), negative control sample BSA.
Wherein L3A monoclonal antibody, the embodiment of the present invention 3 preparation method provided prepares；Rabbit against human T im-3 polyclonal antibody, the embodiment of the present invention 4 preparation method provided prepares.The collocation method of horseradish peroxidase substrate buffer solution is: weigh 10mg3,3 ', 5, and 5 '-tetramethyl benzidine (TMB) is dissolved in 5mL dehydrated alcohol and is prepared as TMB stock solution.Take 0.5mL TMB stock solution time to be used and be added to 10mL phosphate citrate acid substrate buffer solution (0.2mol/L Na2HPO4, 0.1mol/L citric acid) in, add 32 μ L0.75%H2O2Mixing, is configured to horseradish peroxidase substrate buffer solution.Remaining reagent source is with embodiment 1.
2. the using method of test kit
The method that the test kit using the present invention to provide detects people Tim-3 is as follows:
Step 1: being coated monoclonal antibody L3A (10 μ g/mL) in high-affinity elisa plate, 4 DEG C are washed 5 times with the PBS containing 0.2% tween the most afterwards；
Step 2: by suitable dilution factor (e.g., 1: 2～1: 10) dilution normal person or the blood plasma of patient, join in the elisa plate bar being coated with L3A of step 1 gained, hatch 1 hour for 37 DEG C, wash 5 times with the PBS containing 0.2% tween；
Step 3, the polyclonal antibody (1: 5000) of addition rabbit against human T im-3, hatch 1 hour for 37 DEG C, wash 5 times with the PBS containing 0.2% tween；
Step 4, addition HRP labelling goat anti-rabbit antibody (1: 1000), after 37 DEG C hatch 0.5 hour, wash 5 times with the PBS containing 0.2% tween；Adding TMB colour developing, the sulphuric acid of 1mol/L detects the value of OD450 after terminating.
Step 5, judge the concentration of people Tim-3 contained in the blood plasma of patient according to testing result.
Embodiment 7 test kit sensitivity technique
1. material
The preparation method that test kit provides for embodiment 6 prepares.Other reagent are commercial.
2. method
Experiment sets experimental group and matched group, and experimental group albumen to be detected is protein standard substance recombined human Tim-3, and matched group albumen to be detected is Trx albumen.Between different groups, in addition to protein sample difference to be detected, other are completely the same, and concrete operations are as follows:
Step 1: being coated monoclonal antibody L3A in high-affinity elisa plate, 4 DEG C are washed 5 times with the PBS containing 0.2% tween the most afterwards；
Step 2: take protein standard substance recombined human Tim-3 albumen, it is configured to different concentration (concentration is respectively 0.0001ng/mL, 0.001ng/mL, 0.01ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL and 10000ng/mL), join in the elisa plate bar being coated with L3A of step 1 gained, hatch 1 hour for 37 DEG C, wash 5 times with the PBS containing 0.2% tween；
Step 3: add the polyclonal antibody of rabbit against human T im-3, hatch 1 hour for 37 DEG C, washs 5 times with the PBS containing 0.2% tween；
Step 4: add HRP labelling goat anti-rabbit antibody, after 37 DEG C hatch 0.5 hour, washs 5 times with the PBS containing 0.2% tween；Adding TMB colour developing, the sulphuric acid of 1mol/L detects the value of OD450 after terminating.
Step 5: do figure with OD450 value and protein concentration, it is judged that the level of people Tim-3 contained in biological sample.
Testing result is shown in Fig. 2, it is analyzed understanding to it, along with the increase of protein concentration in experimental group, OD450 value is gradually increased, and the OD450 value of all groups of matched group is the most relatively low, and do not change with protein concentration change, illustrate that test kit that the present invention provides can the content of people Tim-3 in specific detection biological sample, its detection is limited to 0.1ng/mL to 1000ng/mL, compared with the minimum detectability 1ng/mL of the people Tim-3 reported, significantly improve detection limit and the sensitivity of people's Tim-3 detection kit.
The level of people Tim-3 in embodiment 8 test kit detection biological specimen
1. material
Test kit is the test kit that embodiment 6 prepares；Normal human serum comes from 10 normal persons；Asthmatic patient serum comes from 10 asthmatic patients of hospital；Type i diabetes patients serum comes from 10 type i diabetes patients of hospital；The serum of Patients with SLE comes from 6 patients of PLA General Hospital systemic lupus erythematosus (sle)；Other reagent are commercially available.
2. method and result
Experiment is divided into negative control, normal human serum group, asthmatic patient serum group, type i diabetes patients serum's group and Serum in Patients with SLE group.Between different groups, in addition to biological sample difference to be detected, other are completely the same, and concrete operations are as follows:
Step 1: being coated monoclonal antibody L3A in high-affinity elisa plate, labelling respectively, 4 DEG C are washed 5 times with the PBS containing 0.2% tween the most afterwards；
Step 2: take normal human serum, asthmatic patient serum, type i diabetes patients serum and Serum in Patients with SLE, join in the elisa plate bar being coated with L3A of step 1 gained, hatch 1 hour for 37 DEG C, wash 5 times with the PBS containing 0.2% tween；
Step 3: add the polyclonal antibody of rabbit against human T im-3, hatch 1 hour for 37 DEG C, washs 5 times with the PBS containing 0.2% tween；
Step 4: add HRP labelling goat anti-rabbit antibody, after 37 DEG C hatch 0.5 hour, washs 5 times with the PBS containing 0.2% tween；Adding TMB colour developing, the sulphuric acid of 1mol/L detects the value of OD450 after terminating.
Step 5: add up the testing result of each group gained, is analyzed summing up.
Test result indicate that negative control absorption value is the lowest, illustrate that kit reagent is normal, data are reliable.The testing result of each group gained of the present embodiment is shown in Fig. 3, according to Fig. 3, compared with adult normal human serum, in type i diabetes patients serum, the content of soluble T im-3 (sTim-3) significantly reduces, compared with soluble T im-3 in Children Normal human serum, in asthmatic children patients serum, the content of soluble T im-3 (sTim-3) significantly raises；In asthmatic patient serum, Serum in Patients with SLE, Tim-3 occurs in that change in various degree, consistent with research report；These all illustrate that the test kit that the present invention prepares can be applied to scientific research and Clinical detection.
The level of people Tim-3 in embodiment 9 test kit detection biological specimen
1. material
Test kit is the test kit that embodiment 6 prepares；The serum of the patient in different sepsis stages: inflammatory syndrome patient (its clinical disease is lighter than sepsis patient) serum comes from 10 patients of hospital；Sepsis patient serum comes from 10 patients of hospital；Patients with severe sepsis serum comes from 10 patients of hospital；The serum of septic shock patient comes from 10 patients of PLA General Hospital；Other reagent are commercially available.
2. method and result
Experiment is divided into inflammatory syndrome group, sepsis group, severe sepsis group and septic shock group.Each group is in addition to biological sample difference to be measured, and other are completely the same, and concrete operations are as follows:
Step 1: being coated monoclonal antibody L3A in high-affinity elisa plate, labelling respectively, 4 DEG C are washed 5 times with the PBS containing 0.2% tween the most afterwards；
Step 2: take inflammatory syndrome serum group, sepsis serum group, severe sepsis serum group and septic shock serum, joins in the elisa plate bar being coated with L3A of step 1 gained, hatches 1 hour for 37 DEG C, washs 5 times with the PBS containing 0.2% tween；
Step 3: add the polyclonal antibody of rabbit against human T im-3, hatch 1 hour for 37 DEG C, washs 5 times with the PBS containing 0.2% tween；
Step 4: add HRP labelling goat anti-rabbit antibody, after 37 DEG C hatch 0.5 hour, washs 5 times with the PBS containing 0.2% tween；Adding TMB colour developing, the sulphuric acid of 1mol/L detects the value of OD450 after terminating.
Step 5: add up the testing result of each group gained, is analyzed summing up.
Experimental result is shown in Fig. 4, can show that in different sepsis Phase patient's serum, the content of soluble T im-3 (sTim-3) is different from figure, and its content increases along with the increase of disease severity, consistent with research report.Illustrate that our test kit can be used for scientific research and Disease Clinical monitoring.
Below being only the preferred embodiment of the present invention, it is noted that above-mentioned preferred implementation is not construed as limitation of the present invention, protection scope of the present invention should be as the criterion with claim limited range.For those skilled in the art, without departing from the spirit and scope of the present invention, it is also possible to make some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (8)
1. the monoclonal antibody of an anti-human Tim-3, it is characterised in that it includes light chain CDR1, light chain
CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3；
The aminoacid sequence of described light chain CDR1 is as shown in SEQ ID NO:1；
The aminoacid sequence of described light chain CDR2 is as shown in SEQ ID NO:2；
The aminoacid sequence of described light chain CDR3 is as shown in SEQ ID NO:3；
The aminoacid sequence of described heavy chain CDR1 is as shown in SEQ ID NO:4；
The aminoacid sequence of described heavy chain CDR2 is as shown in SEQ ID NO:5；
The aminoacid sequence of described heavy chain CDR3 is as shown in SEQ ID NO:6.
Monoclonal antibody the most according to claim 1, it is characterised in that the amino of its variable region of light chain
Acid sequence is as shown in SEQ ID NO:7.
Monoclonal antibody the most according to claim 1, it is characterised in that the amino of its variable region of heavy chain
Acid sequence is as shown in SEQ ID NO:8.
4. the DNA molecular encoding monoclonal antibody as claimed in claim 2, it is characterised in that
It has nucleotide sequence shown in SEQ ID NO:9.
5. the DNA molecular encoding monoclonal antibody as claimed in claim 3, it is characterised in that
It has nucleotide sequence shown in SEQ ID NO:10.
6. the monoclonal antibody as described in claims 1 to 3 any one is used for detecting people Tim-3 in preparation
Immune detection instrument in application.
Application the most according to claim 6, it is characterised in that described immune detection instrument be test kit,
Chip or reagent paper.
8. the test kit containing the monoclonal antibody described in claim 1 to 3 any one.
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| WO2016068803A1 (en) * | 2014-10-27 | 2016-05-06 | Agency For Science, Technology And Research | Anti-tim-3 antibodies |
| GB201419094D0 (en) * | 2014-10-27 | 2014-12-10 | Agency Science Tech & Res | Anti-TIM-3-antibodies |
| CN104592388B (en) * | 2015-03-02 | 2017-05-31 | 中国人民解放军总医院 | A kind of antigen-binding portion thereof of the monoclonal antibody of anti-human Tim 3 |
| EA039020B1 (en) | 2016-04-12 | 2021-11-23 | Симфоген А/С | Anti-tim-3 antibodies and compositions |
| CN105785051B (en) * | 2016-05-05 | 2017-05-31 | 中国人民解放军军事医学科学院基础医学研究所 | In serum the albumen of soluble T im 3 as diagnosing chronic ephrosis mark |
| CN106632675A (en) * | 2016-12-15 | 2017-05-10 | 常州格露康生物医药科技有限公司 | Anti-human Tim-3 monoclonal antibody 8E11 and preparation method thereof |
| CN110621698B (en) | 2017-04-05 | 2024-04-12 | 法国施维雅药厂 | Combination therapies targeting PD-1, TIM-3 and LAG-3 |
| CN108794630A (en) * | 2017-12-18 | 2018-11-13 | 镇江爱必梦生物科技有限公司 | Mouse-anti-human T IM3 protein monoclonal antibodies prepare and its immunohistochemistry purposes |
| CN109266731A (en) * | 2018-08-23 | 2019-01-25 | 窦帅杰 | The interaction of Tim-3 and NF90 is preventing or is treating the application in virus infection product |
| JP7366411B2 (en) * | 2020-01-23 | 2023-10-23 | 国立大学法人北海道大学 | Methods and kits for detecting human α-defensin HD5, and antibodies used therein |
| CN112961239B (en) * | 2021-02-24 | 2021-09-10 | 北京昭衍生物技术有限公司 | TIM inhibitors and uses thereof |
| CN114478778B (en) * | 2022-04-01 | 2022-06-28 | 中国人民解放军军事科学院军事医学研究院 | anti-Tim-3 nano antibody and application thereof |
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