CN111100199B - Fluorescein labeled protein tetramer and preparation method and application thereof - Google Patents
Fluorescein labeled protein tetramer and preparation method and application thereof Download PDFInfo
- Publication number
- CN111100199B CN111100199B CN201911389489.5A CN201911389489A CN111100199B CN 111100199 B CN111100199 B CN 111100199B CN 201911389489 A CN201911389489 A CN 201911389489A CN 111100199 B CN111100199 B CN 111100199B
- Authority
- CN
- China
- Prior art keywords
- protein
- labeled
- fluorescein
- tetramer
- biotin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70521—CD28, CD152
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70521—CD28, CD152
Abstract
The invention provides a fluorescein labeled protein tetramer and a preparation method and application thereof. The fluorescein-labeled protein tetramer is formed by binding fluorescein-labeled streptavidin to a biotin-labeled target protein. The biotin-labeled target protein is expressed in a HEK293 cell in a recombination way, an Avi Tag is co-expressed at the N-end or the C-end of the target protein, and then under the action of BirA enzyme, a lysine residue of the Avi Tag is connected with biotin. And mixing and reacting the biotin-labeled target protein and the fluorescein-labeled streptavidin to obtain the fluorescent direct-labeled protein tetramer. The fluorescent direct-labeling protein tetramer can be used for detecting the positive rate of a neutralizing antibody of a target protein of an immunodetection point by flow screening or CAR-T cells, and the detection sensitivity of the fluorescent direct-labeling protein tetramer is greatly improved compared with that of a fluorescent-labeling protein prepared by other methods because streptavidin and biotin play a role in cascade amplification.
Description
Technical Field
The invention relates to a fluorescein-labeled protein tetramer, a preparation method and application thereof, in particular to a fluorescein-labeled protein tetramer, a preparation method thereof and application thereof in screening neutralizing antibodies of immunodetection point proteins or detecting the positive rate of CAR-T cells.
Background
In recent years, Immunotherapy of cancer, as represented by Immune Checkpoint (Immune Checkpoint) inhibitors and CAR-T (clinical Antigen Receptor T-Cell Immunotherapy) therapy, has become the first line treatment of multiple tumors. The surprising effects of PD-1 inhibitors and CAR-T cell therapy in the treatment of a variety of cancers also establishes new positions for immunotherapy in tumor therapy, particularly emerging CAR-T cell therapies, and is honored as a new hope for cancer cure.
Currently approved immunodetection point inhibitors on the market are based mainly on neutralizing antibodies against immunodetection point proteins, such as tecentiq by roche, Bavencio by fevery, Imfinzi by astrazen, etc. are antibody-based drugs against immunodetection point protein PD-L1, keyruda by MSD, Opdive by BMS, Libtayo by regenerants, etc. are antibody drugs against protein PD1, and yrevo by BMS is antibody drug against protein CTLA-4. The most common screening method used in the development of such antibodies is to flow test their role in the binding of ligand proteins to receptor proteins. However, the interaction between ligand proteins and receptor proteins is generally weak, so the detection window for screening neutralizing antibodies is small.
The principle of CAR-T cell therapy is to modify human T cells by genetic engineering means to express Chimeric Antigen Receptors (CAR) on the cell membrane, and after the CAR is combined with target proteins on the surface of tumor cells, the T cells are activated, so that the specific killing effect of the T cells on the tumors is enhanced. Expression of CAR on the surface of T cells is an indicator that CAR-T cell production must be examined. Conventional detection of CAR expression can select protein L protein and anti-Fab fragment antibody, but protein L only recognizes kappa light chain type CAR, anti-Fab fragment antibody only recognizes Fab structure of CAR, even if CAR expression is detected, it cannot be detected whether CAR on T cell membrane surface can recognize its target antigen. The expression of transfected CAR was most efficient to detect only the corresponding target antigen of CAR, but the detection sensitivity of the CAR-T target protein using monomers was poor, especially in the PK assay for CAR-T therapy.
Disclosure of Invention
It is an object of the present invention to provide a fluorescein labeled protein tetramer.
Another object of the present invention is to provide a method for preparing a fluorescein-labeled protein tetramer.
Another object of the present invention is to provide the use of a fluorescein labeled protein tetramer.
In one aspect, the invention provides a fluorescein-labeled protein tetramer formed by binding fluorescein-labeled streptavidin to a biotin-labeled target protein. The structure of the device is schematically shown in figure 1.
Compared with monomeric protein, the protein tetramer has the characteristics of simple and convenient operation, time saving, high sensitivity and the like when being applied to flow detection, and a principle comparison schematic diagram is shown in figure 2.
According to the specific embodiment of the invention, in the fluorescein-labeled protein tetramer, a biotin-labeled target protein is expressed by HEK293 cells in a recombination way, an Avi Tag is co-expressed at the N-terminal or the C-terminal of the biotin-labeled target protein, and under the action of BirA enzyme, a lysine residue of the Avi Tag is connected with biotin, so that the biotin-labeled target protein is obtained.
According to the specific embodiment of the invention, the tetramer protein comprises two types, wherein one type is fluorescein-labeled protein tetramer of immunodetection point type and is mainly used for establishing a method for screening neutralizing antibody of immunodetection point protein with high sensitivity and simple operation, and the other type is fluorescein-labeled protein tetramer of CAR-T target point type and is mainly used for establishing a high-sensitivity and high-specificity CAR-T cell positive rate detection method. In other words, in the fluorescein-labeled protein tetramer of the present invention, the biotin-labeled target protein is mainly an immunodetection point protein or a CAR-T target protein; preferably, the immunodetection point protein comprises one or more of a series of proteins such as PD1, PD-L1, CD80, CD86, CD28, CTLA-4, CD155, FGL1, LAG3, TIGIT, CD30L, CD30, CD40, CD4L, OX40, OX40L and the like; the CAR-T target protein comprises one or more of a series of proteins such as CD19, BCMA, MSLN, CD22, GCP3, CD20, PMSA and the like.
According to a specific embodiment of the present invention, in the fluorescein-labeled protein tetramer of the present invention, a biotin-labeled target protein is obtained according to the following preparation method: connecting the AviTag gene to the N-end or the C-end of a target protein gene fragment, constructing a recombinant expression vector of the AviTag gene, then co-transfecting the vector plasmid and the BirA enzyme plasmid into a HEK293 cell, adding biotin (biotin) into a cell culture medium, collecting a sample, purifying, and obtaining the biotin-labeled target protein. More specifically, the recombinant expression vector for constructing the target protein is constructed by constructing a target protein gene and an AviTag gene on a carrier pcDNA3.1; preferably, the vector used for constructing the BirA enzyme expression vector is also pcdna3.1.
According to a specific embodiment of the present invention, in the fluorescein-labeled protein tetramer of the present invention, the fluorescein-labeled streptavidin may be commercially available fluorescein-labeled streptavidin, for example, fluorescein includes one or more of FITC, PE, APC, Alexa Flour dye and other commonly used fluorescein.
In another aspect, the present invention also provides a method for preparing the fluorescein-labeled protein tetramer, comprising the steps of: it includes: and mixing the target protein marked by the biotin and the streptavidin marked by the fluorescein according to a ratio, and reacting at room temperature in a dark place to obtain the protein tetramer marked by the fluorescein.
According to a specific embodiment of the present invention, the method for preparing a fluorescein-labeled protein tetramer according to the present invention comprises:
mixing biotinylated target protein and commercialized fluorescein-labeled streptavidin according to a molar ratio of 4:1, fully and uniformly mixing, and reacting for 30 minutes at room temperature in a dark place to obtain a fluorescein-labeled protein tetramer; the obtained fluorescein labeled protein tetramer can be subpackaged, freeze-dried and stored in a refrigerator at minus 80 ℃ for later use.
According to a specific embodiment of the present invention, the method for preparing a fluorescein-labeled protein tetramer according to the present invention further comprises preparing a biotin-labeled protein:
constructing an expression vector containing a target Protein gene and an Avi Tag gene, amplifying and expressing the vector plasmid after sequencing is correct, co-transfecting the vector plasmid and a BirA enzyme plasmid into HEK293 cells, culturing for 6-10 days at 37 ℃, harvesting cell culture supernatant, performing Protein A affinity purification, and desalting by G25 to obtain the purified biotin labeled Protein.
According to a specific embodiment of the present invention, the purified biotin-labeled protein can be replaced with phosphate buffered saline (PBS, pH7.2), and lyophilized for storage.
In another aspect, the present invention further provides a lyophilized powder comprising the fluorescein-labeled protein tetramer of the present invention, wherein the raw materials comprise the fluorescein-labeled protein tetramer of the present invention and a lyophilization solution, wherein the lyophilization solution is Phosphate Buffered Saline (PBS) and contains 0.5% Bovine Serum Albumin (BSA) and 10% Trehalose (Trehalose).
On the other hand, the invention also provides the application of the fluorescein labeled protein tetramer in screening neutralizing antibodies of immunodetection point proteins. The membrane surface of the cell strain expresses ligand proteins of target proteins, fluorescein-labeled protein tetramers can be combined with the ligand proteins, cells are labeled with fluorescein, the fluorescence intensity of the cells is detected through flow, neutralizing antibodies against the ligand proteins or the target proteins are added, and the fluorescence intensity of the cells and the concentration gradient of the added neutralizing antibodies form a negative correlation descending trend.
In a specific class of embodiments of the invention, the biotin-labeled target protein is an immunodetection-type target protein, such as PD1, SIRP-alpha, CD80, CD86, CD155, and the like, and such a fluorescein-labeled protein tetramer can be used to screen for neutralizing antibodies against the immunodetection-point target protein. The design principle of the method of the invention is shown in figure 3: ligand protein of target protein is expressed on the surface of cell membrane, target protein tetramer marked by fluorescein can be combined with the ligand protein, so that corresponding fluorescein is marked on cells, and corresponding fluorescence signals can be detected by flow; if neutralizing antibodies against the ligand protein are added to the experimental system, the antibodies are combined with the ligand protein on the cell membrane, so that the fluorescent signal of the cells detected by flow can be weakened or not detected, and the stronger the affinity of the neutralizing antibodies with the ligand protein, the weaker the fluorescent signal is detected. Similarly, if neutralizing antibodies against the target protein are added, these antibodies will bind to the target protein and will no longer bind to the cell membrane surface ligand protein, and the flow-detected fluorescent signal of the cell will also decrease.
In another aspect, the invention also provides application of the fluorescein labeled protein tetramer in flow detection of CAR-T cell positive rate. The surface of the CAR-T cell membrane expresses a CAR specific to a target protein, a fluorescein-labeled protein tetramer can be specifically bound with the CAR, the CAR-T cell is labeled with fluorescein, and the fluorescence intensity of the cell is detected by flow. Expression level of CAR on CAR-T cell membrane surface is positively correlated with positive rate and fluorescence intensity of cells
In another specific embodiment of the invention, the biotin-labeled target protein is a CAR-T target-like protein. Such as CD19, MSLN, CD22, HER2, BCMA and the like, and the fluorescent labeled protein tetramer can be used for detecting the positive rate of the CAR-T cells. The design principle of the method of the invention is shown in figure 4: the surface of the CAR-T cell membrane expresses a target-specific CAR, a fluorescein-labeled CAR-T target protein tetramer can be specifically bound with the target-specific CAR, and then the CAR-T cell is labeled with fluorescein, so that the fluorescence intensity of the CAR-T cell can be detected in a flow mode. If the CAR expressed by the CAR-T cell to be tested does not recognize the target protein or the CAR is not expressed on the membrane surface, it cannot be labeled with fluorescence and thus no fluorescence signal can be detected.
In conclusion, the invention provides a protein tetramer, a preparation method thereof and application thereof in screening neutralizing antibodies of immunodetection point proteins and detecting positive rate of CAR-T cells. The streptavidin and the biotin play a role in cascade amplification, and the detection sensitivity of the streptavidin and the biotin is greatly improved compared with the fluorescein-labeled protein prepared by other methods.
Drawings
FIG. 1 is a schematic diagram of the preparation of tetrameric protein.
FIG. 2 is a schematic diagram of the principle of the tetrameric protein applied to flow detection.
FIG. 3 is a schematic diagram of the principle of screening for neutralizing antibodies against immunodetection point proteins.
FIG. 4 is a schematic diagram of the CAR-T positive rate detection principle.
FIG. 5 shows that PDCD1/PD 1C-Fc-avi-PE tetramer is capable of enhancing flow detection signal.
FIG. 6 is a histogram showing that flow detection of PDCD1/PD 1C-Fc-avi-PE tetramer recognizes 293T-PD-L1 cell membrane surface PD-L1 protein.
FIG. 7 is a density chart showing that flow-assay PD1/PDCD 1C-Fc-avi-PE tetramer recognizes 293T-PD-L1 cell membrane surface PD-L1 protein.
FIG. 8 shows the application of PD1/PDCD 1C-Fc-avi-PE tetramer to screening anti-PD-L1 neutralizing antibody.
FIG. 9 shows that the CD19Fc-Avi-PE tetramer recognizes the RC222 cell membrane surface expressed CAR.
FIG. 10 shows that CD19Fc-Avi-PE tetramer can enhance flow detection signal.
FIG. 11 shows the application of CD19Fc-Avi-PE tetramer to detect CAR positivity.
Detailed Description
For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description of the embodiments of the present invention taken in conjunction with the accompanying drawings, which are included to illustrate and not to limit the scope of the present invention. In the examples, the experimental methods in which specific conditions are not noted are conventional methods and conventional conditions well known in the art, or conditions as recommended by the instrument manufacturer.
Example 1 preparation and application of immunodetection Point protein tetramer (PDCD1/PD 1C-Fc-avi-PE tetramer)
Preparation of Biotinylated Human PD-1/PDCD1Avi Tag protein
1.1. Reagent and apparatus
TransT1 competent cells (Whole gold, CD 501-01); TransT1 competent cells (ThermoFisher, C505003); tryptone (Sigma, T7293-1KG), yeast extract (Sigma, Y1625-1 KG); agar (Sigma, A7002-1 KG); max plasma Kit (promega, A2492); bacterial shaker (ouruo, HN 211B); bacterial incubator (Tianjin Tester, DH3600B) Nanodrop 2000; expi293F cells (Invitrogen, Cat # R790-07); cell expression culture medium, laboratory self-prepared; polyethylene imine, PEI (Polysciences, Cat # 23966). Cell shaking incubator (Oeno, HNY-2102)
1.2. Solution preparation
LB medium: 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding water to a constant volume of 1000ml, sterilizing under high pressure, cooling to room temperature, adding Amp, and keeping the final concentration at 100ug/ml for later use.
LB solid medium: 5g of tryptone, 2.5g of yeast extract, 5g of sodium chloride and 7.5g of agar, adding water to a constant volume of 500ml, sterilizing under high pressure or boiling in a microwave oven, cooling, adding Amp (the final concentration is 100ug/ml), spreading in an ultra-clean bench, and storing at 4 ℃ for later use.
1.2.3.Polyethylenimine (PEI), Polysciences, Cat #23966,1mg/mL 50mg of PEI powder was dissolved in 45mL of DDH2O, the pH value was adjusted to < 2.0 with HCl, stirring was carried out until PEI was completely dissolved, the pH value was adjusted to 7.0 with NaOH, DDH2O was added to a constant volume of 50mL, 0.22um was filtered and sterilized with a filter, and the mixture was split into 1.0mL portions per tube and frozen at-80 ℃ for use.
1.2.4 preparation of Biotinylated Human PD-1/PDCD1 Avitag
1.3. Construction of expression vectors
5'-CACagtcgcaaggccaccATGCAGATCCCACAGGCGCCC(SEQ ID No.1)
5'-ATAGGGCCCTCTAGATTATTCATGCCATTCGATCTTTTGAG(SEQ ID No.2)
Self signal peptide, expression interval: (21Pro-167 Gln), using cDNA plasmid as a template, HiFI DNA Polymerase, pre-denaturing at 98 ℃ for 3min, denaturing at 98 ℃ for 10s, annealing at 64 ℃ for 30s, extending at 72 ℃ for 30s, repeating for 35 cycles, cutting gel by agarose gel electrophoresis to recover a target fragment, and recombining and connecting to an expression vector in ACRO under the action of a seamless connection kit.
1.4. Plasmid identification and amplification
1.4.1. And (3) plasmid identification: thawing frozen TransT1 competent cells, adding ligation product, mixing, ice-cooling for 30min, heat-shocking at 42 deg.C for 45S, ice-cooling for 2min, adding 700ul LB liquid medium, culturing at 37 deg.C and 200rpm for 1 hr; coating bacteria, and culturing overnight at 37 ℃; picking a single colony the next day, inoculating the single colony into a bacterium shaking tube containing 3ml of LB culture medium, and culturing the single colony overnight in a bacterium shaking table at 37 ℃ at 200 rpm; collecting bacterial liquid, and extracting plasmid DNA according to plasmid Kit instruction of promega; and quantifying Nanodrop2000, sending the obtained product to a sequencing company for sequencing, and checking whether the gene sequence is correct.
1.4.2. And (3) plasmid amplification: the plasmid with the correct sequence was transformed into Top10 strain by the method of 1.4.1, and the plasmid was extracted for cell transfection.
Biotinylated Human PD-1/PDCD1Avi Tag protein expression
1.5.1. Expi293F cells were counted, cell density and viability (cell viability. gtoreq.90%) were calculated, and cell density was adjusted to 1 x 106And/ml, inoculating the cells into a cell culture triangular flask.
1.5.2. Respectively taking corresponding amounts of BirA enzyme plasmids and target gene plasmids and PEI, mixing uniformly, incubating for 5min at room temperature, and adding corresponding volumes into a cell culture triangular flask;
1.5.3. and (3) placing the transfected cells into a shaking incubator for culturing at 37 ℃ and 8% of carbon dioxide for 7 days, collecting cell suspension, centrifuging, and collecting cell culture supernatant for protein purification.
1.6 purification of Biotinylated Human PD-1/PDCD1Avi Tag protein:
after harvesting, the supernatant was collected by centrifugation, purified by ProtinA affinity, and the sample was collected, desalted by G25, and stored in a replacement buffer (PBS containing 10% trehalase) in PBS buffer containing a protective agent (10% Trehalose).
Preparation of PDCD1/PD 1C-Fc-avi-PE tetramer
2.1. Reagent and apparatus
Biotinylated Human PD-1/PDCD1,Avi Tag(AvitagTM) (ii) a PE-Streptavidin (Jackson, Cat. No. 016-110-); 5% BSA-PBS stock solution; 30% of Trehalose-PBS mother liquor; 15ml sterile centrifuge tubes (TrueLine, cat. No. tr2010);a 50ml sterile centrifuge tube (TrueLine, cat. No. tr2012); pipettes (ryinin); penicillin bottles (schottky xinkang pharmaceutical packaging limited, zhong bo 260); lyophilizers (Telstar, lyobeta 5 PS); vortex mixer (Jiangsu health medical products, Inc., model XH-B); super clean bench (Cat. No. DL-CJ-2, Beijing Dong gang Harr instruments manufacturing Co., Ltd.)ND)。
Preparation of PDCD1/PD 1C-Fc-avi-PE tetramer
2.2.1. Preparing a Biotinylated Human PD-1/PDCD1Avi Tag protein solution, slowly dropwise adding a PE-Streptavidin (Jackson, Cat.No. 016-110-. The final molar ratio of Biotinylated Human PD-1/PDCD1, His Tag & Fc Tag, Avi Tag to PE-Streptavidin was 4: 1.
2.2.2. Mixing and standing at 37 ℃ for fully reacting for 30min to obtain the PD-1/PDCD1C-Fc-avi-PE tetramer conjugate. The PD-1/PDCD1C-Fc-avi-PE tetramer conjugate is subpackaged in penicillin bottles, 50 ul/bottle or 250 mu g/bottle, the penicillin bottles are placed in a freeze dryer to be freeze-dried into dry powder, and the dry powder is stored in a refrigerator at minus 80 ℃ for standby after being covered with a label. A schematic of tetrameric protein formation is shown in FIG. 1.
Flow assay for PD-1/PDCD1C-Fc-avi-PE tetramer
3.1. Reagent and apparatus
Biotinylated Human PD-1/PDCD1,Avi Tag(AvitagTM) (ii) a PE-Streptavidin (Jackson, Cat. No. 016-110-); 293T-PD-L1 cells: a 293T stable cell strain for over-expressing human PD-L1, and self-constructing in a laboratory; phosphate Buffered Saline (PBS) (Hyclone, SH 30256.01); flow detection of sample diluent: PBS buffer containing 2% BSA, pH7.4; cell culture dishes (Thermo Fisher Scientific, Cat. No. 150466); 15ml sterile centrifuge tubes (TrueLine, cat. No. tr2010); a 50ml sterile centrifuge tube (TrueLine, cat. No. tr2012); 1.5ml centrifuge tubes (Axygen, MCT-150-C); a pipette.
LUNA II full-automatic counter (logs, Cat. No. L40001) ultra-cleanWorkbench (Cat. No. DL-CJ-2, Tokyo-Harr instruments manufacturing Co., Ltd.)ND) (ii) a Hunan instrument low-speed centrifuger (Hunan instrument centrifuger Co., Ltd., Cat. No. L-550); inverted microscope (OLYMPUS, cat. No. ckx31sf); carbon dioxide incubator (Thermo Fisher Scientific, cat. No. Thermo 3111); flow cytometry (BD, FACSCelestar); flow analysis software (FCS Express 6Flow Research edition).
3.2. The experimental steps are as follows:
3.2.1. collecting cells: the 293T-PD-L1 cell culture supernatant was aspirated, cells were digested with 0.25% trypsin, harvested by centrifugation (1000rpm, 4min, RT), washed 1 time with cell wash buffer, and resuspended in sample diluent.
3.2.2. Cell subpackaging: the cell suspension was mixed well before packaging, according to 1X 10 per sample6The individual cells were packed into centrifuge tubes and washed 1 time for use.
3.2.3. Dilution of tetramer sample: the PD-1/PDCD1C-Fc-avi-PE tetramer was reconstituted with an equal volume of deionized water as before lyophilization, and then diluted with a series of concentration gradients using flow-through sample diluent for the PD-1/PDCD1C-Fc-avi-PE tetramer.
3.2.4. Control sample dilution Biotinylated Human PD-1/PDCD1, His Tag & Fc Tag, Avi Tag (Acro, Cat. NO. PD1-H82F2, Lot. NO. BV1052-74DF1-EV) protein was diluted with flow sample diluent to the same concentration gradient as PD-1/PDCD1C-Fc-Avi-PE tetramer.
3.2.5. Reacting with cells: adding the diluted sample and 100 ul/tube into each tube of cells, blowing, mixing, and incubating at 4 deg.C for 1 h.
3.2.6. Incubation of PE-Streptavidin: after the cells of the control sample group were incubated with Biotinylated Human PD-1/PDCD1, His Tag & Fc Tag, Avi Tag, 1:200 diluted PE-Streptavidin was added and incubated for 1h at 4 ℃. The tetramer experimental group did not require this step.
3.2.7. And (3) washing cells: after incubation and 3 washes, 200ul PBS was added to resuspend the cells and transferred to a flow loading tube for use.
3.2.8. Flow detection: detecting the corresponding fluorescent signals respectively by using the corresponding fluorescent detection channels; and storing the FCS format file copy on a computer equipped with De Novo Software-FCS Express 6Flow Research Edition streaming analysis Software for data analysis.
3.3. The experimental results are as follows:
analysis of the results showed that: the PDCD1/PD 1C-Fc-avi-PE tetramer protein has a detection signal in flow detection which is far stronger than that of a monomeric protein Biotinylated Human PD-1/PDCD1 control group, and the MFI is increased by 10-20 times (see Table 1). At low concentration (1ug/m), the positive rate of the Biotinylated Human PD-1/PDCD1 control group was reduced to 49.68%, while the positive rate of PDCD1/PD 1C-Fc-avi-PE was not reduced (98.82%); the PDCD1/PD 1C-Fc-avi-PE tetramer can enhance the detection signal when used for flow detection, and the results are shown in FIG. 5, FIG. 6 and FIG. 7.
TABLE 1 flow assay results for PD-1/PDCD1C-Fc-avi-PE tetramers
4. Application case for screening neutralizing antibody of anti-PD-L1
4.1. Reagent and apparatus
293T-PD-L1 cells: a 293T stable cell strain for over-expressing human PD-L1, and self-constructing in a laboratory; PD-1/PDCD1C-Fc-avi-PE tetramer; anti-human PD-L1 antibody, produced by laboratory; phosphate Buffered Saline (PBS) (Hyclone, SH 30256.01); flow detection of sample diluent: PBS buffer containing 2% BSA, pH7.4; cell culture dishes (Thermo Fisher Scientific, Cat. No. 150466); 15ml sterile centrifuge tubes (TrueLine, cat. No. tr2010); a 50ml sterile centrifuge tube (TrueLine, cat. No. tr2012); 1.5ml centrifuge tubes (Axygen, MCT-150-C); liquid transfer device
LUNA II full-automatic counter (logs, Cat. No. L40001) super clean bench (Cat. No. DL-CJ-2, manufactured by Beijing Toyobo Harr instruments Co., Ltd.)ND) (ii) a Hunan instrument low-speed centrifuger (Hunan instrument centrifuger Co., Ltd., Cat. No. L-550); inverted microscope (OLYMPU)S, cat.no. ckx31sf); carbon dioxide incubator (Thermo Fisher Scientific, cat. No. Thermo 3111); flow cytometry (BD, FACSCelestar); flow analysis software (FCS Express 6Flow Research edition).
4.2. The experimental steps are as follows:
4.2.1. collecting cells: the 293T-PD-L1 cell culture supernatant was aspirated, cells were digested with 0.25% trypsin, harvested by centrifugation (1000rpm, 4min, RT), washed 1 time with cell wash buffer, and resuspended in sample diluent.
4.2.2. Cell subpackaging: the cell suspension was mixed well before packaging, according to 1X 10 per sample6The individual cells were packed into centrifuge tubes and washed 1 time for use.
4.2.3. Dilution of tetramer sample: the PD-1/PDCD1C-Fc-avi-PE tetramer was reconstituted with an equal volume of deionized water as before lyophilization and then diluted to 0.2ug/ml with the flow-through sample diluent for PD-1/PDCD1C-Fc-avi-PE tetramer.
4.2.4. anti-PD-L1 antibody dilution: anti-PD-L1 antibody was diluted with a flow sample diluent for a series of concentration gradients.
4.2.5. Mixing PD-1/PDCD1C-Fc-avi-PE tetramer and anti-PD-L1 antibody in the same volume, adding the mixture into a centrifugal tube containing cells after uniformly mixing, blowing and uniformly mixing the mixture in 100 ul/tube, and standing the mixture at 4 ℃ for incubation for 1 h.
4.2.6. Flow detection: detecting the corresponding fluorescent signals respectively by using the corresponding fluorescent detection channels; and storing the FCS format file copy on a computer equipped with De Novo Software-FCS Express 6Flow Research Edition streaming analysis Software for data analysis.
4.3. The experimental results are as follows:
the PDCD1/PD 1C-Fc-avi-PE tetramer protein is used for screening the neutralizing antibody of the antibody human PD-L1. The experimental results are shown in fig. 8, the PDCD1/PD 1C-Fc-avi-PE protein tetramer is combined with 293T-PD-L1 cells to generate the fluorescence signal of PE, but the fluorescence signal of PE detected by flow is weaker and weaker with the increase of the dosage of the added anti-human PD-L1 neutralizing antibody (the fluorescence intensity is shown in table 2).
TABLE 2 flow assay results of neutralizing antibodies screening for antibody human PD-L1
| Neutralizing antibody of antibody human PD-L1 | Average fluorescence intensity (MFI) of PE |
| 10.00ug/ml | 44. |
| 3.00ug/ml | 42. |
| 2.00ug/ml | 44. |
| 1.00ug/ml | 48. |
| 0.50ug/ml | 288. |
| 0.30ug/ml | 1337. |
| 0.10ug/ml | 2605. |
| 0.03ug/ml | 2842. |
| 0.00ug/ml | 2966. |
Example 2 preparation and application of CAR-T target protein tetramer (CD19 Fc-Avi-PE tetramer)
Preparation of Biotinylated Human CD19 FcAvi Tag protein
1.1. Reagent and apparatus
TransT1 competent cells (Whole gold, CD 501-01); TransT1 competent cells (ThermoFisher, C505003); tryptone (Sigma, T7293-1KG), yeast extract (Sigma, Y1625-1 KG); agar (Sigma, A7002-1 KG); max plasma Kit (promega, A2492); bacterial shaker (ouruo, HN 211B); bacterial incubator (Tianjin Tester, DH3600B) Nanodrop 2000; expi293F cells (Invitrogen, Cat # R790-07); cell expression culture medium, laboratory self-prepared; polyethylene imine, PEI (Polysciences, Cat # 23966). Cell shaker incubators (Ono, HNY-2102).
1.2. Solution preparation
LB medium: 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding water to a constant volume of 1000ml, sterilizing under high pressure, cooling to room temperature, adding Amp, and keeping the final concentration at 100ug/ml for later use.
LB solid medium: 5g of tryptone, 2.5g of yeast extract, 5g of sodium chloride and 7.5g of agar, adding water to a constant volume of 500ml, sterilizing under high pressure or boiling in a microwave oven, cooling, adding Amp (the final concentration is 100ug/ml), spreading in an ultra-clean bench, and storing at 4 ℃ for later use.
1.2.3.Polyethylenimine (PEI), Polysciences, Cat #23966,1mg/mL 50mg of PEI powder was dissolved in 45mL of DDH2O, the pH value was adjusted to < 2.0 with HCl, stirring was carried out until PEI was completely dissolved, the pH value was adjusted to 7.0 with NaOH, DDH2O was added to a constant volume of 50mL, 0.22um was filtered and sterilized with a filter, and the mixture was split into 1.0mL portions per tube and frozen at-80 ℃ for use.
1.3. Construction of expression vectors
5'-GGGGCGGCCGCGCCACCATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCCCGAGGAACCTCTAGTG-3'(SEQ ID No.3)
5'-GGGACGCGTTTATTCATGCCATTCGATCTTTTG-3'(SEQ ID No.4)
Self signal peptide, expression interval: (20Pro-291Lys), using cDNA plasmid as a template, using HiFI DNA Polymerase, pre-denaturing at 98 ℃ for 3min, denaturing at 98 ℃ for 10s, annealing at 64 ℃ for 30s, extending at 72 ℃ for 30s, performing 35 cycles, performing agarose gel electrophoresis, cutting gel, recovering a target fragment, and performing recombinant connection to an expression vector in ACRO under the action of a seamless connection kit.
1.4. Plasmid identification and amplification
1.4.1. And (3) plasmid identification: thawing frozen TransT1 competent cells, adding ligation product, mixing, ice-cooling for 30min, heat-shocking at 42 deg.C for 45S, ice-cooling for 2min, adding 700ul LB liquid medium, culturing at 37 deg.C and 200rpm for 1 hr; coating bacteria, and culturing overnight at 37 ℃; picking a single colony the next day, inoculating the single colony into a bacterium shaking tube containing 3ml of LB culture medium, and culturing the single colony overnight in a bacterium shaking table at 37 ℃ at 200 rpm; collecting bacterial liquid, and extracting plasmid DNA according to plasmid Kit instruction of promega; and quantifying Nanodrop2000, sending the obtained product to a sequencing company for sequencing, and checking whether the gene sequence is correct.
1.4.2. And (3) plasmid amplification: the plasmid with the correct sequence was transformed into Top10 strain by the method of 1.4.1, and the plasmid was extracted for cell transfection.
Biotinylated Human CD19Fc Avi Tag protein expression
1.5.1. The cells of Expi293F were counted, the cell density and the cell viability (cell viability. gtoreq.90%) were calculated, and the cell density was adjusted to 1 x 106/ml and inoculated into a cell culture flask.
1.5.2. Respectively taking corresponding amounts of BirA enzyme plasmids and target gene plasmids and PEI, mixing uniformly, incubating for 5min at room temperature, and adding corresponding volumes into a cell culture triangular flask;
1.5.3. and (3) placing the transfected cells into a shaking incubator for culturing at 37 ℃ and 8% of carbon dioxide for 7 days, collecting cell suspension, centrifuging, and collecting cell culture supernatant for protein purification.
Biotinylated Human CD19Fc Avi Tag protein purification:
after harvesting, the supernatant was collected by centrifugation, purified by Protin a affinity purification, and the sample was collected, desalted by G25, and stored in a replacement buffer (PBS containing 10% trehalase) in PBS buffer containing a protective agent (10% Trehalose).
Preparation of Biotinylated Human CD19Fc Avi Tag tetramer
2.1. Reagent and apparatus
Biotinylated Human CD19Fc Avi Tag; PE-Streptavidin (Jackson, Cat. No. 016-110-); 5% BSA-PBS stock solution; 30% of Trehalose-PBS mother liquor; 15ml sterile centrifuge tubes (TrueLine, cat. No. tr2010); a 50ml sterile centrifuge tube (TrueLine, cat. No. tr2012); pipettes (ryinin); penicillin bottles (schottky xinkang pharmaceutical packaging limited, zhong bo 260); lyophilizers (Telstar, lyobeta 5 PS); vortex mixer (Jiangsu health medical products, Inc., model XH-B); super clean bench (Cat. No. DL-CJ-2, Beijing Dong gang Harr instruments manufacturing Co., Ltd.)ND)。
Preparation of CD19Fc-Avi-PE tetramer
2.2.1. Preparing a Biotinylated Human CD19Fc Avi Tag protein solution, slowly dripping PE-Streptavidin (Jackson, Cat.No. 016-110-. The final molar ratio of Biotinylated Human CD19Fc Avi Tag to PE-Streptavidin was 4: 1.
2.2.2. Mixing and standing at 37 ℃ for fully reacting for 30min to obtain the CD19Fc-Avi-PE tetramer conjugate. The CD19Fc-Avi-PE tetramer conjugate is subpackaged in penicillin bottles, 50 ul/bottle or 250 mu g/bottle, the penicillin bottles are placed in a freeze dryer to be freeze-dried into dry powder, and the dry powder is stored in a refrigerator at minus 80 ℃ for standby after being covered with a label. A schematic of tetrameric protein formation is shown in FIG. 1.
Flow-based identification of CD19Fc-Avi-PE tetramer
3.1. Reagent and apparatus
Biotinylated Human CD19Fc Avi Tag; PE-Streptavidin (Jackson, Cat. No. 016-110-); RC222 cell line (membrane surface over-expressed CAR, FCM63-scFv), laboratory self-constructed; phosphate Buffered Saline (PBS) (Hyclone, SH 30256.01); flow detection of sample diluent: PBS buffer containing 2% BSA, pH7.4; cell culture dishes (Thermo Fisher Scientific, Cat. No. 150466); 15ml sterile centrifuge tubes (TrueLine, cat. No. tr2010); a 50ml sterile centrifuge tube (TrueLine, cat. No. tr2012); 1.5ml centrifuge tubes (Axygen, MCT-150-C); a pipette.
LUNA II full-automatic counter (logs, Cat. No. L40001) super clean bench (Cat. No. DL-CJ-2, manufactured by Beijing Toyobo Harr instruments Co., Ltd.)ND) (ii) a Hunan instrument low-speed centrifuger (Hunan instrument centrifuger Co., Ltd., Cat. No. L-550); inverted microscope (OLYMPUS, cat. No. ckx31sf); carbon dioxide incubator (Thermo Fisher Scientific, cat. No. Thermo 3111); flow cytometry (BD, FACSCelestar); flow analysis software (FCS Express 6Flow Research edition).
3.2. The experimental steps are as follows:
3.2.1. collecting cells: the RC222 cell culture supernatant was aspirated, cells were digested by addition of 0.25% trypsin, harvested by centrifugation (1000rpm, 4min, RT), washed 1 time with cell washing buffer, and cells resuspended in sample diluent.
3.2.2. Cell subpackaging: the cell suspension was mixed well before packaging, according to 1X 10 per sample6The individual cells were packed into centrifuge tubes and washed 1 time for use.
3.2.3. Dilution of tetramer sample: the CD19Fc-Avi-PE tetramer was reconstituted with an equal volume of deionized water as before lyophilization, and the CD19Fc-Avi-PE tetramer was diluted with a series of concentration gradients using flow sample diluent.
3.2.4. Control sample dilution the Biotinylated Human CD19Fc Avi Tag protein was diluted with the same concentration gradient using flow sample diluent.
3.2.5. Reacting with cells: adding the diluted sample and 100 ul/tube into each tube of cells, blowing, mixing, and incubating at 4 deg.C for 1 h.
3.2.6. Incubation of PE-Streptavidin: after the cells of the control sample group were incubated with Biotinylated Human CD19Fc Avi Tag, 1:200 dilution of PE-Streptavidin was added and incubated at 4 ℃ for 1 h. The tetramer experimental group did not require this step.
3.2.7. And (3) washing cells: after incubation and 3 washes, 200ul PBS was added to resuspend the cells and transferred to a flow loading tube for use.
3.2.8. Flow detection: detecting the corresponding fluorescent signals respectively by using the corresponding fluorescent detection channels; and storing the FCS format file copy on a computer equipped with De Novo Software-FCS Express 6Flow Research Edition streaming analysis Software for data analysis.
3.3. The experimental results are as follows:
the cell membrane surface of RC222 expresses CAR capable of binding with CD19 protein, and the flow detection result shows that CD19Fc-Avi-PE tetramer can bind with RC222 cells, while the PE-Streptavidin control group does not detect the binding with RC222 cells. And the detection signal of the CD19Fc-Avi-PE tetramer is far stronger than that of a monomeric protein Biotinylated Human CD19Fc Avi Tag control group, and specific detection data are shown in Table 3, figure 9 and figure 10.
TABLE 3 flow assay results for PD-1/PDCD1C-Fc-avi-PE tetramers
Case of D19Fc-Avi-PE tetramer for CAR-T Positive Rate detection
4.1. Reagent and apparatus
Biotinylated Human CD19Fc Avi Tag; PE-Streptavidin (Jackson, Cat. No. 016-110-); tetramer negative control protein (Acrobiosystems, cat. No. la3-HP2H 3); HEK293 cells; RC222 cell line (membrane surface over-expressed CAR, FCM63-scFv), laboratory self-constructed; phosphate Buffered Saline (PBS) (Hyclone, SH 30256.01); flow detection of sample diluent: PBS buffer containing 2% BSA, pH7.4; cell culture dishes (Thermo Fisher Scientific, Cat. No. 150466); 15ml sterile centrifuge tubes (TrueLine, cat. No. tr2010); a 50ml sterile centrifuge tube (TrueLine, cat. No. tr2012); 1.5ml centrifuge tubes (Axygen, MCT-150-C); a pipette.
LUNA II full-automatic counter (logs, Cat. No. L40001) super clean bench (Cat. No. DL-CJ-2, manufactured by Beijing Toyobo Harr instruments Co., Ltd.)ND) (ii) a Hunan instrument low-speed centrifuger (Hunan instrument centrifuger Co., Ltd., Cat. No. L-550); inverted microscope (OLYMPUS, cat. No. ckx31sf); carbon dioxide incubator (Thermo Fisher Scientific, cat. No. Thermo 3111); flow cytometry (BD, FACSCelestar); flow analysis software (FCS Express 6Flow Research edition).
4.2. The experimental steps are as follows:
4.2.1. collecting cells: HEK293 cells and RC222 cells were separately pipetted off the supernatant, cells were digested by addition of 0.25% trypsin, harvested by centrifugation (1000rpm, 4min, RT), washed 1 time with cell wash buffer, and resuspended in sample diluent.
4.2.2. Cell subpackaging: the cell suspension was mixed well before packaging, according to 1X 10 per sample6The individual cells were packed into centrifuge tubes and washed 1 time for use.
4.2.3. Dilution of tetramer sample: the CD19Fc-Avi-PE tetramer and tetramer negative control protein were reconstituted separately with an equal volume of deionized water as before lyophilization and diluted to 1ug/ml separately with flow sample diluent (secondary concentrations determined by experimental optimization).
4.2.4. Reacting with cells: adding the diluted sample and 100 ul/tube into each tube of cells, blowing, mixing, and incubating at 4 deg.C for 1 h.
4.2.5. And (3) washing cells: after incubation and 3 washes, 200ul PBS was added to resuspend the cells and transferred to a flow loading tube for use.
4.2.6. Flow detection: detecting the corresponding fluorescent signals respectively by using the corresponding fluorescent detection channels; and storing the FCS format file copy on a computer equipped with De Novo Software-FCS Express 6Flow Research Edition streaming analysis Software for data analysis.
4.3. The experimental results are as follows:
the membrane surface of RC222 cells overexpresses CARs capable of recognizing CD19, as CAR-like T cells in this application, HEK293 cells were not transfected with CD19, as negative control cells in this case. The experimental results showed that the CD19Fc-Avi-PE tetramer was able to bind to RC222 cell membrane without binding to the empty 293 cells, while the negative control tetramer protein was not bound to RC222 (see FIG. 11 for details). The CD19Fc-Avi-PE tetramer can specifically recognize the CAR of CD19, and can be used for detecting the positive rate of CAR-T.
Sequence listing
<110> Beijing Baipusais Biotech Co., Ltd
<120> fluorescein labeled protein tetramer and preparation method and application thereof
<130> GAI19CN5952
<150> CN 201811640376.3
<151> 2018-12-29
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 39
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 1
cacagtcgca aggccaccat gcagatccca caggcgccc 39
<210> 2
<211> 41
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 2
atagggccct ctagattatt catgccattc gatcttttga g 41
<210> 3
<211> 92
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 3
ggggcggccg cgccaccatg ccacctcctc gcctcctctt cttcctcctc ttcctcaccc 60
ccatggaagt caggcccgag gaacctctag tg 92
<210> 4
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223> primer
<400> 4
gggacgcgtt tattcatgcc attcgatctt ttg 33
Claims (8)
1. Use of a fluorescein-labeled protein tetramer in flow-assay for CAR-T cell positivity, wherein the fluorescein-labeled protein tetramer is formed by binding fluorescein-labeled streptavidin to a biotin-labeled target protein, the biotin-labeled target protein is a CAR-T target protein, and the CAR-T target protein is CD 19.
2. The use according to claim 1, wherein the biotin-labeled target protein is recombinantly expressed by HEK293 cells, an Avi Tag is co-expressed at the N-terminus or the C-terminus of the target protein, and a lysine residue of the Avi Tag is linked with biotin under the action of the BirA enzyme, so as to obtain the biotin-labeled target protein.
3. The use according to claim 2, wherein the biotin-labeled target protein is obtained by the following preparation method: connecting the AviTag gene to the N-end or the C-end of a target protein gene fragment, constructing a recombinant expression vector of the AviTag gene, then co-transfecting the vector plasmid and the BirA enzyme plasmid into a HEK293 cell, adding biotin (biotin) into a cell culture medium, collecting a sample, purifying, and obtaining the biotin-labeled target protein.
4. The use according to claim 3, wherein the recombinant expression vector for constructing the protein of interest is constructed by constructing the gene of the protein of interest and the AviTag gene into the vector pcDNA3.1.
5. The use according to claim 4, wherein the vector used for the construction of the BirA enzyme expression vector is pcDNA3.1.
6. Use according to claim 1, wherein fluorescein is labeled streptavidin and fluorescein comprises one or more of FITC, PE, APC, Alexa Flour dyes.
7. The use according to claim 1, wherein the fluorescein-labeled protein tetramer is prepared according to the following method: and mixing the target protein marked by the biotin and the streptavidin marked by the fluorescein according to a ratio, and reacting at room temperature in a dark place to obtain the protein tetramer marked by the fluorescein.
8. The use according to claim 1, wherein the fluorescein-labeled protein tetramer is in the form of a lyophilized powder, and the raw materials comprise the fluorescein-labeled protein tetramer and a lyophilization solution, wherein the lyophilization solution is Phosphate Buffered Saline (PBS) containing 0.5% Bovine Serum Albumin (BSA) and 10% Trehalose (Trehalose).
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811640376 | 2018-12-29 | ||
| CN2018116403763 | 2018-12-29 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111100199A CN111100199A (en) | 2020-05-05 |
| CN111100199B true CN111100199B (en) | 2021-02-12 |
Family
ID=70424341
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911389489.5A Active CN111100199B (en) | 2018-12-29 | 2019-12-30 | Fluorescein labeled protein tetramer and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111100199B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112552415B (en) * | 2021-02-22 | 2021-06-22 | 北京百普赛斯生物科技股份有限公司 | B lymphocyte stimulating factor dodecamer, and preparation method and application thereof |
| CN113061589A (en) * | 2021-03-17 | 2021-07-02 | 领诺(上海)医药科技有限公司 | Preparation method and application of cell strain for stably expressing biotin ligase Bir A |
| WO2023072006A1 (en) * | 2021-10-25 | 2023-05-04 | 黄�俊 | Use of polymer in detection and preparation of cells expressing car |
| CN116930475A (en) * | 2022-03-31 | 2023-10-24 | 上海细胞治疗集团药物技术有限公司 | Neutralizing antibody detection method |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| HUE050406T2 (en) * | 2014-08-08 | 2020-12-28 | Univ Leland Stanford Junior | High affinity pd-1 agents and methods of use |
| CN105884870B (en) * | 2016-01-25 | 2022-05-27 | 大连大学 | Method for preparing tetramer from O-type foot-and-mouth disease virus polypeptide |
| US11965021B2 (en) * | 2017-04-26 | 2024-04-23 | Eureka Therapeutics, Inc. | Cells expressing chimeric activating receptors and chimeric stimulating receptors and uses thereof |
-
2019
- 2019-12-30 CN CN201911389489.5A patent/CN111100199B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN111100199A (en) | 2020-05-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111100199B (en) | Fluorescein labeled protein tetramer and preparation method and application thereof | |
| CN107843730B (en) | Method for reversibly staining target cells | |
| CN108490174B (en) | Method for detecting CAR-T cells and application thereof | |
| CN108508200B (en) | Method for detecting CD19 CAR-expressing cells and application thereof | |
| EP2972330A2 (en) | Protocol for identifying and isolating antigen-specific b cells and producing antibodies to desired antigens | |
| AU2023248207A1 (en) | Methods and compositions for identifying epitopes | |
| US20120064543A1 (en) | Method for detecting substance in biological sample | |
| CN112433055A (en) | Method for detecting biological activity of PVRIG antibody based on reporter gene method | |
| KR102041119B1 (en) | Reagent for assaying anti-treponema pallidum antibody | |
| US20210071241A1 (en) | In situ cell analysis in cell culture system | |
| CN111826398A (en) | Engineering plasmid system for displaying and detecting interaction of membrane protein between living cells | |
| WO2012159075A1 (en) | Bioprobe compositions and their methods of use | |
| CN112540176B (en) | Kit, method and computer-readable storage medium for diagnosing diseases associated with FAP expression abnormality | |
| US20220411472A1 (en) | Self-assembling circular tandem repeat proteins with increased stability | |
| CN114921496A (en) | Construction method and application of humanized immune system animal model with NK (Natural killer) cell and ADCC (advanced Charge coupled device) capabilities | |
| CN104560911B (en) | A kind of fusion antigen protein matter | |
| CN114487448B (en) | Composition and kit for detecting myasthenia gravis-related antibody and application of composition and kit | |
| CN111393528A (en) | Single-chain antibody targeting folate receptor α and application thereof | |
| CN110423270B (en) | Preparation of Toxoplasma gondii surface antigens GRA1 and GRA7 recombinant protein | |
| CN104558135B (en) | A kind of antigen protein with 65 antibody specific bond of glutamte dehydrogenase | |
| CN115109159A (en) | CD19 mutant protein and application thereof | |
| CN110437341B (en) | Detection protein with red fluorescence activity and application thereof | |
| US20240036027A1 (en) | Nanopore Biosensors and Uses Thereof | |
| WO2023186106A1 (en) | Method for detecting neutralizing antibody | |
| Schofield et al. | Sequencing and affinity determination of antigen-specific B lymphocytes from peripheral blood |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 100176 floor 4, building 4, No. 8, Hongda North Road, Beijing Economic and Technological Development Zone, Daxing District, Beijing Applicant after: Beijing baipusai Biotechnology Co., Ltd Address before: 100176 floor 4, building 4, No. 8, Hongda North Road, Beijing Economic and Technological Development Zone, Daxing District, Beijing Applicant before: BEIJING ACROBIOSYSTEMS Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |