CN114836388A - Claudin18.2 monoclonal antibody, preparation method and application thereof - Google Patents
Claudin18.2 monoclonal antibody, preparation method and application thereof Download PDFInfo
- Publication number
- CN114836388A CN114836388A CN202210637801.3A CN202210637801A CN114836388A CN 114836388 A CN114836388 A CN 114836388A CN 202210637801 A CN202210637801 A CN 202210637801A CN 114836388 A CN114836388 A CN 114836388A
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- immunogen
- polypeptide
- cross
- seq
- 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.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
Abstract
The invention provides a Claudin18.2 monoclonal antibody, a preparation method and application thereof. The invention adopts the general microbiological center preserved in China Committee for culture Collection of microorganisms with the preservation number: the Claudin18.2 monoclonal antibody prepared from the hybridoma cell of CGMCC No.45150 can be used for detecting Claudin18.2 in a sample.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a Claudin18.2 monoclonal antibody and a preparation method and application thereof.
Background
The Claudin18.2 protein is a transmembrane protein located on the cell membrane, and is found to be highly expressed in various tumors, particularly digestive system tumors and metastases thereof. In recent years, the specific antibody of claudiximab (zolbetuximab/IMAB362) has achieved remarkable success in recent clinical trials, and the claudin18.2 protein is expected to become a molecular target for targeted therapy of certain specific malignant tumors. However, there is no murine monoclonal antibody of Claudin18.2 available for mouse and rat samples for Western blot use on the market at present.
Disclosure of Invention
In view of the above disadvantages of the prior art, the present invention aims to provide a Claudin18.2 monoclonal antibody, a preparation method and a use thereof, which fills the blank of the prior art.
The invention provides a hybridoma cell strain HOO57-Claudin 18.2 which is preserved in China general microbiological culture Collection center, and the preservation number is as follows: CGMCC No. 45150.
The invention also provides a Claudin18.2 monoclonal antibody secreted and produced by the hybridoma cell strain HOO57-Claudin 18.2.
Further, the Claudin18.2 monoclonal antibody is IgG 1.
In another aspect, the invention provides the use of a Claudin18.2 monoclonal antibody in the preparation of a formulation for detecting Claudin18.2 in a sample.
Further, the detection is an immunoblotting or immunohistochemical assay.
The invention also provides a kit for detecting Claudin18.2 in a sample, wherein the kit contains the Claudin18.2 monoclonal antibody.
In another aspect, the present invention provides a method for preparing the above Claudin18.2 monoclonal antibody, said method comprising the steps of:
1) providing an immunogen: adopting polypeptide with an amino acid sequence shown as SEQ ID NO.1 as immunogen and crosslinking with carrier protein to obtain crosslinked immunogen;
2) preparing hybridoma cells: immunizing animals by cross-linked immunogen, taking spleen of the immunized animals to fuse with mouse myeloma cells to prepare hybridoma cells and culturing;
3) preparing a monoclonal antibody: the hybridoma cells are injected into the abdominal cavity of a mouse, and the monoclonal antibody is extracted from ascites.
Further, the polypeptide shown in SEQ ID NO.1 is further modified to obtain the polypeptide shown in SEQ ID NO.2, and the polypeptide shown in SEQ ID NO.2 is crosslinked with carrier protein.
Further, the carrier protein is Keyhole Limpet Hemocyanin (KLH) or Bovine Serum Albumin (BSA).
Further, the N end of the cross-linked immunogen is covalently cross-linked with the amino group of the carrier protein through a cross-linking agent.
Furthermore, the cross-linked immunogen and the immune adjuvant are mixed and emulsified, and then are injected subcutaneously at multiple points on the back of a mouse, and the serum titer is more than 1: 20000 after more than two times of boosting.
Further, the method for extracting monoclonal antibodies is the prior art, and a person skilled in the art can know how to extract monoclonal antibodies by the prior knowledge.
Further, the method comprises a step 4) of purifying the monoclonal antibody.
Further, the purification includes protein G affinity purification and peptide affinity purification.
As mentioned above, the Claudin18.2 monoclonal antibody, the preparation method and the application thereof have the following beneficial effects:
the Claudin18.2 monoclonal antibody provided by the invention can effectively identify the Claudin 18.2.
Cell name: hybridoma cell
Preservation time: 04/29/2022
The preservation unit: china general microbiological culture Collection center
The preservation number is: CGMCC No.45150
And (4) storage address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
Drawings
FIG. 1 is a Western blot chart, and the band of about 17kDa detected in lane B is the Claudin18.2 protein signal.
Detailed Description
A polypeptide having the sequence: TQDLYNNPVT (SEQ ID NO. 1). The monoclonal antibody against Claudin18.2 prepared by the polypeptide can specifically recognize natural Claudin18.2 protein in tissues or cells in immunoblotting (Western blot) and Immunohistochemical (IHC) analysis. The anti-Claudin 18.2 antibody is obtained by the following steps:
the method comprises the following steps: analysis and design of polypeptide sequence: the amino acid sequence of the Claudin18.2 protein is subjected to epitope analysis by using Abdesigne software, indexes such as hydrophilicity, antigenicity, surface possibility, flexibility and the like are mainly evaluated, and the 10 amino acids from 32 to 42 positions of the Claudin18.2 protein are finally determined as the amino acid sequence of a synthetic polypeptide by combining with the practical experience of preparing antibodies in the past, wherein the sequence is TQDLYNNPVT (SEQ ID NO. 1).
Step two: polypeptide synthesis and cross-linking: synthesizing target polypeptide by using an ACT396 full-automatic multi-channel polypeptide synthesizer, and identifying by adopting mass spectrum;
to enhance the antigenicity of the polypeptide, the Claudin18.2 polypeptide was crosslinked to the carrier protein KLH using a Sulfo-SMCC crosslinking method.
Step three: polypeptide immunization and hybridoma preparation: mixing and emulsifying the crosslinked Claudin 18.2-KLH and Freund's adjuvant, carrying out intradermal injection immunization on the back of a BalB/C mouse, and repeatedly boosting the immunization until the titer of an antibody detected by blood sampling reaches the standard.
Step four: : preparing a hybridoma: three days after booster immunization, mice were sacrificed, spleens were ground and mixed with mouse myeloma cells in a 10: 1 ratio and PEG fusion. Culturing in HAT and HT culture medium in sequence, obtaining monoclonal by limiting dilution method, detecting OD value of supernatant by indirect ELISA, selecting cell strain of monoclonal cell strain with high OD value, and performing amplification culture.
Step five: preparing ascites: BalB/C mice were injected intraperitoneally with sterile paraffin oil, and 7 days later, the prepared hybridomas were injected intraperitoneally. After the abdomen of the mouse rises for 7 days, the ascites of the mouse is extracted.
Step six: antibody purification: separating ascites from mouse, purifying the antibody with Protein A, and further purifying with peptide affinity to obtain the target antibody.
The anti-Claudin 18.2 monoclonal antibody is identified by the following steps: the obtained Claudin18.2 antibody was used as a primary antibody for detecting an antigen by a standard immunoblotting method, confirming that the antibody can interact with the denatured native Claudin18.2 protein.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art. Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
Examples
1) Analysis and design of polypeptide sequences
The method mainly comprises the steps of performing epitope analysis on the amino acid sequence of the Claudin18.2 protein by using Abdesigne software, mainly evaluating indexes such as hydrophilicity, antigenicity, surface possibility, flexibility and the like, and finally determining 10 amino acids at positions 32-42 of the Claudin18.2 protein as a synthetic polypeptide amino acid sequence with the sequence being TQDLYNNPVT (SEQ ID NO.1) by combining actual experience of preparing antibodies in the past and considering amino acid structure complexity, easy oxidation degree, synthetic difficulty, amino acid types and distribution and the like. Meanwhile, in order to ensure the affinity purification of the later polypeptide cross-linked carrier protein and the peptide, a cysteine C is added at the N terminal, and the sequence of the finally synthesized polypeptide is CTQDLYNNPVT (SEQ ID NO.2)
2) Polypeptide synthesis and crosslinking
Synthesizing: the ACT396 full-automatic multi-channel polypeptide synthesizer automatically synthesizes target polypeptides according to a programmed program, the synthesized polypeptides are dissolved in 50% acetonitrile, a mass spectrometer is adopted for identification, and the obtained polypeptides are confirmed to be the target polypeptides.
And (3) crosslinking: Sulfo-SMCC as a cross-linker cross-linked the carrier protein KLH with the synthetic polypeptide: 10mg of KLH was dissolved in 0.5ml of ultrapure water; 3mg of sulfo-SMCC was dissolved in 0.5ml of ultrapure water, and the pH was adjusted to about 7 with 3M NaOH. In the case of mixing, the sulfo-SMCC solution was slowly added dropwise to the KLH solution, and the reaction was stirred and mixed at room temperature for 30 min. The reacted sulfo-SMCC/KLH mixture was loaded onto a Sephadex G25 column equilibrated in advance with equilibration buffer (0.05M PB, pH6.0) for 30min, and the light gray eluate, i.e., the activated sulfo-SMCC/KLH solution, was collected. 2mg of the crosslinked polypeptide was dissolved in 200. mu.l of PBS (pH7.3), 0.2 volume of the sulfo-SMCC/KLH complex solution was added to the polypeptide solution, the pH was adjusted to 7.3, the mixture was shaken at room temperature for 4 hours, frozen at-70 ℃ and lyophilized in a lyophilizer for 24 hours. Polypeptide crosslinking efficiency is determined by detecting polypeptide sulfydryl before and after crosslinking through an Ellman method, and the coupling rate is 98.4%.
3) Polypeptide immunization and antiserum preparation
100. mu.g of the crosslinked KLH-polypeptide was dissolved in 100. mu.l of phosphate buffer (0.01M PBS) and emulsified well (until no diffusion in water) by adding an equal volume of Freund's complete adjuvant. Healthy BalB/C mice with the age of 6 weeks and the weight of 20-25g are used for immunization, and the immunization is performed by intradermal injection at the back, and at least 3 points are required for injection. After 2 weeks of the first immunization, 100. mu.g of the polypeptide was dissolved in 100. mu.l of phosphate buffer (0.01M PBS), and the resulting solution was thoroughly emulsified with an equal amount of incomplete Freund's adjuvant and then subjected to intradermal immunization, and the immunization was required to be performed by intradermal injection into the back as a first booster immunization at least at 3 points. 2 weeks after the second immunization, a second boost was performed, the method and requirements were identical to the first boost. After 1 week, tail blood is taken, the ELISA plate is coated with non-crosslinked synthetic polypeptide, and the titer of immune serum is detected by an indirect ELISA method. The booster immunization and the titer determination are repeated until the serum titer reaches more than 1: 10000. After the potency reached the requirement, 100ug of polypeptide was dissolved in 0.5ml phosphate buffer (0.01M PBS) and injected intraperitoneally as a booster.
4) Hybridoma cell preparation
(1) Cell fusion: three days after boosting, the eyeballs of the mice were bled and the mice were sacrificed. Soaking in 75% alcohol for sterilization, taking mouse spleen from an ultra-clean bench, grinding with a 200-mesh steel sieve, and adding DMEM to constant volume to 10 ml. The ground spleen was then dispersed into single cells by pipetting. Counting 10ul of the mixed solution, and calculating the total number of splenocytes. Following myeloma cell: the spleen cells were added with the corresponding amount of myeloma cells at 1:10, mixed well, centrifuged at 1000r/min for 5 minutes and the supernatant discarded. The mixed cells were flicked to disperse them loosely and placed in a water bath at 37 ℃. Add 1ml PEG1500 with a pipette while stirring for 1 min. Stirring for 1min, stirring PEG gently, and standing for 90 s. 1ml of serum-free DMEM was added with stirring, PEG was stirred up, and the reaction was stopped. 40ml serum free DMEM was added with stirring, centrifuged at 1000rpm/min for 5min, the PEG washed off and the supernatant discarded.
(2) And (3) cell culture screening: the bottom fused cells were gently blown up with 2ml of fused cell culture solution, and made up to 50ml with HAT culture solution (HAT in DMEM containing 15% calf serum). The cells were evenly distributed into 96-well plates containing feeder cells, 100. mu.l/well. After 5 days, the supernatant in the 96-well plate was discarded and replaced with a newly prepared HAT medium (HAT in DMEM containing 15% calf serum). After 3 days of fluid change, 100ul of supernatant per well was placed on an ELISA plate coated with the antigen, and the positive value of the supernatant was measured by indirect ELISA. And taking the hole with the high positive value, replacing HAT culture solution, and rechecking after one day. The positive wells were subjected to limiting dilution with HT medium (HT in DMEM containing 15% calf serum) and distributed evenly to 96-well plates containing feeder cells at 100. mu.l/well. One week later, the plate positive values were checked, and the monoclonal with high positive values was subcloned in DMEM containing 15% calf serum. After the positive monoclonal subcloning, the positive rate of the pore plate supernatant reaches 100%, the cell strain with high positive value and large cell group area is taken for amplification culture, and the obtained hybridoma cell is sent to the China general microbiological culture Collection center for preservation, with the preservation number: CGMCC No. 45150.
5) Preparation of monoclonal antibodies
One week before cell killing, 3 healthy 8-week-old BalB/C female mice were injected with 1ml of paraffin oil per abdominal cavity. When the cells were in log phase, the cells in the cell vial were counted by blowing down 3 x 10 6 The cells are put into a centrifuge tube and centrifuged for 5min at 1000 r/min. The supernatant was discarded, 5ml of sterile phosphate buffer (0.01M PBS) was added, cells were blown off with a pipette gun, 30ml of phosphate buffer (0.01M PBS) was supplemented, and the mixture was centrifuged at 1000rpm for 5 min. 3ml of phosphate buffer (0.01M PBS) was added to the centrifuge tube and mixed well, and 1ml of the mixture was injected into the abdominal cavity of each mouse. After one week of injection, the neck is cut off to kill the mouse, the mouse is soaked in 75% alcohol for disinfection, the abdominal epithelium is cut off by scissors, the syringe is inserted into the abdominal cavity to pick up the abdominal cavity membrane, the ascites is sucked into a centrifugal tube, and the centrifugal tube is centrifuged for 5min at 3500 r/min. The supernatant was collected, mixed with the binding buffer at 1:1 and filtered through a 0.45um filter.
6) Antibody affinity purification
(1) IgG purification: 10ml of 50% Protein-A Sepharose suspension was applied to a 30ml column using a liquid dispenser, the top and bottom caps were removed, the volume of the column bed after the liquid flow-out was 5ml, and then washed 3 times with 25ml deionized water. 10ml of the corresponding serum was removed, mixed with 2ml of PBS and added to a 30ml column, and the serum sample was allowed to flow out by vortexing on a rotary mixer at room temperature (20-25 ℃) for 1 hour. The column was washed 3 times with 20ml of purified wash solution and eluted with 10ml of eluent.
(2) Peptide affinity purification: 1ml of Sulfo-link gel suspension (0.5ml of gel) was added to the column, and after the column liquid drained, the column was washed with 4ml of coupling buffer. The synthesized Claudin18.2 polypeptide was dissolved in 1ml of coupling buffer and added to the column, and 1ml of coupling buffer was added to the column, and mixed by inversion at room temperature for 1 hour. The column was washed with 6ml of coupling buffer, then 3ml of blocking solution was added and mixed well for 1 hour at room temperature. The column was washed 3 times, then 6ml IgG and 3ml PBS were added to the column, and mixed by inversion at room temperature for 1 hour. The column was washed 3 times with PBS and then eluted with 2ml of eluent. The obtained purified antibody was packed in a dialysis bag and dialyzed at 4 ℃. Dialyzed overnight, and then centrifuged at 4000rpm X35 min to remove the precipitate, and the supernatant was collected. The titer of the antibody measured by an indirect ELISA method is 1: 32000 the protein concentration was determined by the Bradford method and was 1.023 mg/ml.
7) Immunoblot analysis
Preparing SDS-PAGE gel according to a standard method, sequentially adding 5 mul of lysate with the protein concentration of 5mg/ml, keeping the pressure constant for about 30 minutes at 80V, changing the voltage to 160V when a sample runs through the concentrated gel and is basically in a straight line, stopping electrophoresis until a bromophenol blue indicator completely runs out of the separation gel (about 60 minutes), and performing constant-pressure 100V electric conversion to 80 minutes by adopting an electric membrane conversion method to convert the membrane to a PVDF membrane. The obtained Claudin18.2 antibody was used as a primary antibody, and the obtained antibody was hybridized with the antigen obtained by the above-mentioned membrane transfer at room temperature for 1 hour at a concentration of 1.25. mu.g/ml, and then hybridized with an HRP-labeled goat anti-rabbit antibody at room temperature for 1 hour, and color development was carried out by ECL color development, and color development and exposure were carried out by X-ray in a dark room, and the immunoblotting results were as shown in FIG. 1.
The results show that: the prepared Claudin18.2 monoclonal antibody can effectively identify the Claudin 18.2.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (10)
1. A hybridoma cell strain HOO57-Claudin 18.2, which is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is as follows: CGMCC No. 45150.
2. The hybridoma cell line HOO57-Claudin 18.2 of claim 1, which secretes Claudin18.2 monoclonal antibody.
3. Use of the Claudin18.2 monoclonal antibody of claim 2 in the preparation of a formulation for detecting Claudin18.2 in a sample.
4. A kit for detecting Claudin18.2 in a sample, comprising the Claudin18.2 monoclonal antibody of claim 2.
5. The method of preparing the Claudin18.2 monoclonal antibody of claim 4, which comprises the steps of:
1) providing an immunogen: adopting polypeptide with an amino acid sequence shown as SEQ ID NO.1 as immunogen and crosslinking with carrier protein to obtain crosslinked immunogen;
2) preparing hybridoma cells: immunizing animals by cross-linked immunogen, taking spleen of the immunized animals to fuse with mouse myeloma cells to prepare hybridoma cells and culturing;
3) preparing a monoclonal antibody: the hybridoma cells are injected into the abdominal cavity of a mouse, and the monoclonal antibody is extracted from ascites.
6. The method of claim 5, wherein: the polypeptide shown in SEQ ID NO.1 is further modified to obtain the polypeptide shown in SEQ ID NO.2, and the polypeptide shown in SEQ ID NO.2 is crosslinked with carrier protein.
7. The method of claim 5, wherein: the carrier protein is keyhole limpet hemocyanin or bovine serum albumin.
8. The method of claim 5, wherein: the N end of the cross-linked immunogen is subjected to covalent cross-linking of the sulfhydryl group and the amino group of the carrier protein through a cross-linking agent.
9. The method of claim 5, wherein: the cross-linked immunogen and the immune adjuvant are mixed and emulsified, and then are injected subcutaneously at multiple points on the back of a mouse, and the serum titer is more than 1: 20000 after more than two times of boosting immunity.
10. The method of claim 5, wherein: the method comprises the step 4) of purifying the monoclonal antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210637801.3A CN114836388A (en) | 2022-06-07 | 2022-06-07 | Claudin18.2 monoclonal antibody, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210637801.3A CN114836388A (en) | 2022-06-07 | 2022-06-07 | Claudin18.2 monoclonal antibody, preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114836388A true CN114836388A (en) | 2022-08-02 |
Family
ID=82573982
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210637801.3A Pending CN114836388A (en) | 2022-06-07 | 2022-06-07 | Claudin18.2 monoclonal antibody, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114836388A (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2366709A1 (en) * | 2010-03-16 | 2011-09-21 | BioNTech AG | Tumor vaccination involving a humoral immune response against self-proteins |
| US20130052217A1 (en) * | 2010-03-16 | 2013-02-28 | Thorsten Klamp | Tumor vaccination involving a humoral immune response against self-proteins |
| CN103694353A (en) * | 2007-05-29 | 2014-04-02 | 加尼梅德药物公司 | Monoclonal antibodies against claudin-18 for treatment of cancer |
| CN104321345A (en) * | 2012-05-09 | 2015-01-28 | 加尼梅德药物公司 | Antibodies against claudin 18.2 useful in cancer diagnosis |
| WO2020043044A1 (en) * | 2018-08-27 | 2020-03-05 | 南京圣和药业股份有限公司 | Anti-claudin18.2 antibody and use thereof |
| CN112979817A (en) * | 2021-04-27 | 2021-06-18 | 宝船生物医药科技(上海)有限公司 | Monoclonal antibody for recognizing anti-CLDN 18_2 antibody and preparation method and application thereof |
| CN114507287A (en) * | 2022-04-20 | 2022-05-17 | 苏州百道医疗科技有限公司 | anti-CLDN 18.2 recombinant rabbit monoclonal antibody and application thereof |
| CN114560941A (en) * | 2020-11-27 | 2022-05-31 | 广东东阳光药业有限公司 | Antibodies of CLDN18.2 and uses thereof |
-
2022
- 2022-06-07 CN CN202210637801.3A patent/CN114836388A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103694353A (en) * | 2007-05-29 | 2014-04-02 | 加尼梅德药物公司 | Monoclonal antibodies against claudin-18 for treatment of cancer |
| EP2366709A1 (en) * | 2010-03-16 | 2011-09-21 | BioNTech AG | Tumor vaccination involving a humoral immune response against self-proteins |
| US20130052217A1 (en) * | 2010-03-16 | 2013-02-28 | Thorsten Klamp | Tumor vaccination involving a humoral immune response against self-proteins |
| CN104321345A (en) * | 2012-05-09 | 2015-01-28 | 加尼梅德药物公司 | Antibodies against claudin 18.2 useful in cancer diagnosis |
| WO2020043044A1 (en) * | 2018-08-27 | 2020-03-05 | 南京圣和药业股份有限公司 | Anti-claudin18.2 antibody and use thereof |
| CN110862454A (en) * | 2018-08-27 | 2020-03-06 | 南京圣和药业股份有限公司 | anti-Claudin 18_2 antibody and application thereof |
| CN112654638A (en) * | 2018-08-27 | 2021-04-13 | 南京圣和药业股份有限公司 | anti-Claudin18.2 antibody and application thereof |
| CN114560941A (en) * | 2020-11-27 | 2022-05-31 | 广东东阳光药业有限公司 | Antibodies of CLDN18.2 and uses thereof |
| CN112979817A (en) * | 2021-04-27 | 2021-06-18 | 宝船生物医药科技(上海)有限公司 | Monoclonal antibody for recognizing anti-CLDN 18_2 antibody and preparation method and application thereof |
| CN114507287A (en) * | 2022-04-20 | 2022-05-17 | 苏州百道医疗科技有限公司 | anti-CLDN 18.2 recombinant rabbit monoclonal antibody and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| HAISHAN LIN等: "Development of anti-human CLDN18.2 monoclonal antibody as cancer therapeutics", POSTER PRESENTATIONS - PROFFERED ABSTRACTS, vol. 8, no. 4 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4446122A (en) | Purified human prostate antigen | |
| USRE33405E (en) | Purified human prostate antigen | |
| CN111349617B (en) | Hybridoma cell strain secreting anti-Tau or pTau-181/231/396 monoclonal antibody and application thereof | |
| CN87105716A (en) | Utilize insolubilized immune complexes to improve the production of antibody | |
| CN110903389A (en) | Monoclonal antibody and cell line for resisting GFAP protein, and preparation method and application thereof | |
| JP3891542B2 (en) | Methods for generating immunological reactants specific for phosphorylation sites | |
| RU2163243C2 (en) | Goat female liver proteins showing antitumor activity, pharmaceutical composition | |
| CN101880316B (en) | Human RBPMS polypeptide and preparation method of antibody thereof | |
| CN112625135A (en) | Dicofol monoclonal antibody and application thereof | |
| CN112626033B (en) | IRG1 monoclonal antibody and preparation method and application thereof | |
| CN112662632B (en) | ADORA2A-AS1 monoclonal antibody, and preparation method and application thereof | |
| CN114836388A (en) | Claudin18.2 monoclonal antibody, preparation method and application thereof | |
| CN108148127A (en) | A kind of people MAP3K7IP2 polypeptides and its preparation method for antibody | |
| CN114957438B (en) | Human Abeta 1-42 epitope polypeptide for detecting Alzheimer disease and preparation method thereof | |
| CN114075281B (en) | anti-Inhibin-alpha protein monoclonal antibody, cell line, preparation method and application thereof | |
| WO1981001849A1 (en) | Purified human prostate antigen | |
| CN114807052A (en) | SIGLEC15 monoclonal antibody, preparation method and application thereof | |
| CN101928334B (en) | Preparation method of mouse Ehf polypeptide and antibody thereof | |
| CN111187754A (en) | Hybridoma cell strain, anti-trichina intestinal serine protease monoclonal antibody produced by hybridoma cell strain and application of anti-trichina intestinal serine protease monoclonal antibody | |
| CN114437210B (en) | Polypeptide for preparing rice AGO16 protein antibody, and preparation method and application thereof | |
| CN113845595B (en) | anti-GH protein monoclonal antibody, cell line, preparation method and application thereof | |
| LU101593B1 (en) | Nilaparvata lugens PCE3 polypeptide and method for preparing polyclonal antibody thereof | |
| CN111349170B (en) | Monoclonal antibody of immune related GTPase family M (IRGM) and application thereof | |
| CN117264031B (en) | Group B neisseria meningitidis fHBP recombinant protein and application thereof | |
| CN110054674B (en) | Immunogenic polypeptide, anti-TTC 36 antibody AP2-19 and application |
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 |