CN109851673B - Preparation method of anti-human B7-H3 monoclonal antibody, immunohistochemical detection method and application thereof, and kit thereof - Google Patents
Preparation method of anti-human B7-H3 monoclonal antibody, immunohistochemical detection method and application thereof, and kit thereof Download PDFInfo
- Publication number
- CN109851673B CN109851673B CN201910058619.0A CN201910058619A CN109851673B CN 109851673 B CN109851673 B CN 109851673B CN 201910058619 A CN201910058619 A CN 201910058619A CN 109851673 B CN109851673 B CN 109851673B
- Authority
- CN
- China
- Prior art keywords
- human
- monoclonal antibody
- chain variable
- variable region
- light chain
- 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
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
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Oncology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hospice & Palliative Care (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses an anti-human B7-H3 monoclonal antibody, which comprises a heavy chain and a light chain, wherein the amino acid sequence of a heavy chain variable region (mVH) is the same as that of SEQ ID NO.1, and the specific steps are as follows: EVQLVESGGGLVKPGGSLKLSCAASGFTFSRYAMSWVRQTPEKRLEWVATISDDGRNTYDRDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCVRHRAITTARFDYWGQGTSLTVSS; the amino acid sequence of the light chain variable region (mVL) is the same as SEQ ID NO.2, and is specifically as follows: DILMTQSPSSMSVSLGDTVSITCHASQDISSNIGWLQQGPGNSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISGLESEDFADYYTLQSVQFPYTFGGGTKLEIK. The invention also discloses a detection method, application and a kit thereof. The anti-human B7-H3 monoclonal antibody provided by the invention can be used for immunohistochemical detection of tumor tissues, improves the accuracy of the immunohistochemical detection of tumor specimens, is beneficial to diagnosis of malignant tumors, determination of primary parts and pathological typing, and improves the diagnosis accuracy of tumors, especially low-differentiation or undifferentiated tumors.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of an anti-human B7-H3 monoclonal antibody, an immunohistochemical detection method, application and a kit thereof.
Background
In recent years, more and more researches find that B7-H3 molecules are abnormally expressed in various tumor tissues, such as ovarian cancer, breast cancer, lung cancer, kidney cancer, colorectal cancer, prostate cancer and the like. Clinical retrospective disease pattern analysis found that expression of B7-H3 was correlated with clinical pathology, survival analysis found that cases with high expression of B7-H3 had poor prognosis, high mortality, and high levels of soluble B7-H3 (sB 7-H3) were also found in peripheral blood of tumor patients. In addition, B7-H3 has been found to be associated with graft immune rejection and autoimmune pathology, and grafts transfected with the B7-H3 gene can inhibit the activity of host T cells, thereby prolonging the survival time of the graft. The research results indicate that B7-H3 is an important negative costimulatory molecule and has important research and application values.
In many human tumors, B7-H3 is expressed at both mRNA and protein levels and is inversely related to the prognosis of the patient. At present, various tumors have been studied retrospectively in a few documents, and the functions of B7-H3 in anti-tumor immunity are analyzed from the clinical point of view, wherein the functions include ovarian cancer, esophageal cancer, renal cancer, gastric cancer, liver cancer, lung cancer, colon cancer, pancreatic cancer, breast cancer, prostate cancer and melanoma.
B7—h3 is abnormally expressed in tumor tissue or peripheral blood of tumor patients and is closely related to age, pathology type, tumor biological effect and patient survival rate.
Therefore, the development of the mouse anti-human B7-H3 monoclonal antibody can lay a material foundation for further researching the action of the molecule in tumors, and simultaneously provides an effective research means for exploring B7-H3 signal transduction paths and mechanisms, and the functional anti-human B7-H3 monoclonal antibody is more hopeful to become a potential target point for tumor biological treatment.
Disclosure of Invention
In order to overcome the defects in the prior art, one of the purposes of the invention is to provide an anti-human B7-H3 monoclonal antibody.
Another object of the present invention is to provide a method for detecting the heavy chain and light chain variable regions of an anti-human B7-H3 antibody.
The invention also aims to provide an application of the anti-human B7-H3 monoclonal antibody.
The fourth object of the invention is to provide a kit for anti-human B7-H3 monoclonal antibody.
In order to achieve one of the purposes of the invention, the technical scheme adopted is as follows:
an anti-human B7-H3 monoclonal antibody comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain variable region (mVH) is identical to SEQ ID No.1, and is specifically as follows:
EVQLVESGGGLVKPGGSLKLSCAASGFTFSRYAMSWVRQTPEKRLEWVATISDDGRNTYDRDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCVRHRAITTARFDYWGQGTSLTVSS；
the amino acid sequence of the light chain variable region (mVL) is the same as SEQ ID NO.2, and is specifically as follows:
DILMTQSPSSMSVSLGDTVSITCHASQDISSNIGWLQQGPGNSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISGLESEDFADYYTLQSVQFPYTFGGGTKLEIK。
in a preferred embodiment of the invention, the heavy chain variable region (mVH) has three hypervariable regions: GFTFSRYA, ISDDGRNT, VRHRAITTARFDY.
In a preferred embodiment of the invention, the light chain variable region (mVL) has three hypervariable regions: QDISSN, HGT, LQSVQFPYT.
In order to achieve the second object of the invention, the technical scheme adopted is as follows:
a method for detecting anti-human B7-H3 antibodies in the heavy and light chain variable regions comprising the steps of:
(1) Preparing hybridoma of B7-H3 monoclonal antibody;
(1.1) immunizing a mouse;
(1.2) cell culture;
(1.3) fusion and screening;
(2) Preparing a B7-H3 mouse/human chimeric antibody;
(2.1) extracting cDNA of the hybridoma cells: extracting RNA from the hybridoma cell strain, and reversely transcribing the obtained RNA into cDNA by using an RT-PCR technology;
cloning the heavy chain variable region (mVH) and the light chain variable region (mVL) of the hybridoma cells by using specifically designed upstream and downstream primers; the sequences of the specifically designed upstream and downstream primers are nucleotide SEQ ID NO.3 and nucleotide sequence 4 respectively;
(2.2) the heavy chain variable region (mVH) and the light chain variable region (mVL) are respectively connected with a cloning vector (pJET cloning vector), the connection product is used for transforming competent bacteria DH5a, and the pJET vector carries an ampicillin (amp+) resistance gene, so that the transformed bacteria can be smeared on an Amp-resistant LB solid medium and cultured overnight at 37 ℃;
(2.3) bacteria to be coated grow into scattered colonies, and selecting the colonies with clear edges and good growth, and further sequencing and identifying;
(2.4) reserving candidate heavy chain variable region (mVH) and light chain variable region (mVL) sequences according to the sequencing result, amplifying heavy chain variable region (mVH) and light chain variable region (mVL) sequences matched with the expression vector by PCR again, connecting the PCR product with a linear expression vector which is subjected to double enzyme digestion pretreatment, transforming competent bacteria DH5a by the connection product, coating transformed bacteria liquid on Kana resistant LB solid medium due to the fact that the expression vector carries kanamycin (Kana+) resistance genes, and culturing at 37 ℃ overnight;
(2.5) comparing the sequencing results of the two times according to the sequencing method (2.4), selecting the transformant with the correct sequence, and carrying out plasmid extraction after amplification culture;
(2.6) cotransfecting the eukaryotic expression cell line 293 with an expression vector having the genes of the desired monoclonal antibody heavy chain variable region (mVH) and light chain variable region (mVL) linked thereto;
(2.7) the supernatant obtained contains the target antibody, the flow detection expression monoclonal antibody is well combined with M435, and the positive rate is more than 95%, which shows that the correct antibody heavy chain and light chain variable region sequences are obtained.
In a preferred embodiment of the present invention, wherein, in step (2.6), the eukaryotic expression cell line 293 is suspension cultured, SFM4Transfx-293 witout L-glutamine (liquid) serum-free medium is passaged and expanded and replaced with Gibco, freeStyle ™ 293 Expression Medium serum-free medium at the time of transfection; the supernatant was harvested after 7 days of continuous culture, centrifuged at 4000g for 30min, and the supernatant was freed from impurities such as cells and the like and sterilized by filtration with a 0.45um filter.
In order to achieve the third object of the present invention, the technical scheme adopted is as follows:
an application of an anti-human B7-H3 monoclonal antibody, wherein the anti-human B7-H3 monoclonal antibody is used for preparing a kit for immunohistochemical detection of tumor tissues.
In order to achieve the fourth object of the invention, the technical scheme adopted is as follows:
a kit of anti-human B7-H3 monoclonal antibodies, wherein the kit comprises the above-described anti-human B7-H3 monoclonal antibodies.
In addition, the invention also provides an anti-human B7-H3 monoclonal antibody which is used for immunohistochemical detection of tissue specimens, and the results of detecting colorectal cancer and gastric cancer tissue specimens by using the antibody B7-H3 show that the B7-H3 is not basically expressed in normal colorectal tissues and gastritis tissues, the expression of the B7-H3 is up-regulated in a neoplastic stage, and the B7-H3 is highly expressed in colorectal cancer and gastric cancer, and is related to prognosis and survival of patients.
The invention has the beneficial effects that:
the anti-human B7-H3 monoclonal antibody provided by the invention can be used for immunohistochemical detection of tumor tissues, improves the accuracy of the immunohistochemical detection of tumor specimens, is beneficial to diagnosis of malignant tumors, determination of primary parts and pathological typing, and improves the diagnosis accuracy of tumors, especially low-differentiation or undifferentiated tumors.
Drawings
FIG. 1a is a schematic diagram of flow cytometry analysis of positive control (positive control) of anti-human B7-H3 mab 4H12 to L929/B7-H3 surface B7-H3 molecules.
FIG. 1B is a schematic diagram of a flow cytometry analysis of the anti-human B7-H3 mab 4H12 versus the L929/B7-H3 surface B7-H3 molecule.
FIG. 2 is a schematic representation of B7-H3 antibodies for immunohistochemical detection of colorectal cancer tissue.
FIG. 3a is a schematic diagram showing abnormal expression of B7-H3 at different stages of colorectal cancer occurrence and development (1).
FIG. 3B is a schematic diagram showing abnormal expression of B7-H3 at different stages of colorectal cancer occurrence and development (2).
FIG. 4a is a schematic diagram showing the results of histochemical staining of B7-H3 mab against superficial gastritis, atrophic gastritis, low grade neoplasia, high grade neoplasia and gastric cancer tissue (1).
FIG. 4B is a schematic diagram showing the results of histochemical staining of B7-H3 mab against superficial gastritis, atrophic gastritis, low grade neoplasia, high grade neoplasia and gastric cancer tissue (2).
FIG. 5 is a schematic diagram showing the results of immunohistochemical detection of B7-H3 molecules performed on 160 clinical gastric cancer tissues.
Detailed Description
The invention will now be further described with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The examples are all routine experimentation in the art unless otherwise specified.
The explanation of fig. 1-5 is as follows:
FIG. 1 a/1B shows the recognition of the B7-H3 molecule on the L929/B7-H3 surface by flow cytometry analysis of anti-human B7-H3 mab 4H12, wherein the gray peak is a negative control.
A is a positive control (positive control), and the primary antibody is a commercial PE-labeled anti-human B7-H3 monoclonal antibody.
B is an experimental group, the primary antibody is an anti-human B7-H3 antibody 4H12, and the secondary antibody is fluorescein PE marked goat anti-human IgG.
Control, commercial B7-H3 antibody positive Control;
4H 12. The anti-human B7-H3 monoclonal antibody of the invention;
count is the number of cells and cooo-FL 2 is the PE fluorescence intensity.
FIG. 2 shows the use of B7-H3 antibodies for immunohistochemical detection of colorectal cancer tissue.
FIG. 3a shows the abnormal expression of B7-H3 at various stages of colorectal cancer development.
B7-H3 on A is not expressed in normal intestinal epithelium;
under A, B, C, B7-H3 expression is gradually up-regulated in the development process of intraepithelial neoplasia, inflammation and colorectal cancer.
FIG. 3B shows the expression level of B7-H3 during the evolution of colorectal cancer, which is not substantially expressed in normal colorectal tissue, and which is up-regulated in colorectal adenoma stage, from polyps, adenomas, high grade tumors to carcinomas, suggesting that the expression of B7-H3 molecules during different stages of colorectal cancer development and progression may be involved in regulating tumor immune responses at different phases.
FIG. 4 a-4B shows that B7-H3 is not substantially expressed in inflammatory tissues, and is up-regulated in the stage of tumor transformation, and B7-H3 is abnormally and highly expressed in gastric cancer tissues when B7-H3 monoclonal antibody is used for carrying out histochemical staining on superficial gastritis, atrophic gastritis, low-grade neoplasia, high-grade neoplasia and gastric cancer tissues (from left to right).
FIG. 5 shows that the prognosis results of patients with B7-H3 molecule immunohistochemical detection on 160 clinical gastric cancer tissues and CD8 positive cell analysis of negative co-stimulatory molecules B7-H3 combined infiltration show that B7-H3/CD8 is of a certain significance for clinical medication guidance and prognosis evaluation of gastric cancer patients, and in tissues with low CD8 infiltration B7-H3 high expression, the prognosis of patients is poor, while the prognosis of patients with high CD8 expression is good.
The sources of the biological materials used in the examples are as follows:
example 1 acquisition of B7-H3 monoclonal antibody-producing hybridomas
(one) immunized mice
Mice were immunized with the fusion protein or transgenic cells four times, 21 days apart, and the orbital blood titers of the mice were measured 7-10 times after the fourth immunization, and the immunization was boosted after the titers were measured.
(II) cell culture
1.1 d before fusion, 1 BALB/c mouse with the age of 6-7 weeks is taken and placed in 75% ethanol solution for 2min.
And 2, taking out the spleen of the mouse aseptically, placing the spleen in a 200-mesh stainless steel screen, and grinding to obtain single cell suspension. The cells were washed twice (1400 rpm,5 min) with 1640 basal medium for further use. Cell concentration was adjusted to 2X 10 with 1640 medium containing 15% FBS 5 Per ml, drop into 96 well culture plates at 100. Mu.L per well, 37℃and 5% CO 2 Culturing in an incubator.
3. Overnight incubation, the next day was observed under low power microscope. And 150ul of subcloned cells were plated.
(III) fusion and screening
Preparation:
1. and (3) equipment: a mouse soaking cup; a simple dissecting table; a hybridoma pack; a heat preservation water bath cup; a thermometer; 96-well culture plates.
2. Reagent: 75% ethanol solution; preheating 1640 basal medium at 37 ℃; preheating 1ml of PEG at 37 ℃; 14ml of basal medium (PEG terminator) was pre-warmed 1640 at 37 ℃; the selection medium (containing 15% FBS and the amount of HAT added was calculated such that the final HAT concentration of medium in the final 96-well plate was 1%) was pre-warmed 1640 at 37 ℃.
The steps are as follows:
1. the immunized mice were rinsed with running water and placed in 75% ethanol solution for 2min.
2. The spleens of the mice were removed aseptically, placed in a 200 mesh stainless steel screen, and ground to obtain a single cell suspension. The medium was washed twice (1400 rpm,5 min) with pre-warmed 1640 basal medium for further use.
3. SP2/0 cells well grown in the logarithmic growth phase were collected and washed twice (1400 rpm,5 min) with pre-warmed 1640 basal medium for later use.
4. SP2/0 cells or Ag8 cells and spleen cells were mixed in a 50ml clear plastic centrifuge tube, typically at a ratio of 5:1 spleen cells to myeloma cells, pre-heated 1640 basal medium was washed once (1400 rpm,5 min), the supernatant was discarded (to avoid unnecessary dilution of PEG) and the bottom of the tube was rubbed with the palm (or gently flicked with a finger) to thoroughly mix the two cells into suspended cells.
5. The centrifuge tube is placed in a 37 ℃ water-bath cup for preheating, 1ml of 50% PEG solution preheated at 37 ℃ is sucked, the centrifuge tube is added at a constant speed within 1min, the centrifuge tube is gently shaken while the centrifuge tube is added, and the centrifuge tube is gently shaken for 60s in the 37 ℃ water bath after the centrifuge tube is added. (3 seconds drop by drop)
6. The addition of 14ml of 1640 basal medium pre-warmed at 37℃along the tube wall was stopped (1 min 1ml 3min 3ml, finally 10 ml was slowly added). (dripping at a constant speed)
After resting at 7.37℃for 5min, it was centrifuged (800 rpm,5 min) and the supernatant was discarded (the tube was tilted and the supernatant was aspirated).
8. The precipitated cells were gently resuspended (not bearable) with 1640 selection medium preheated at 37℃and added to the preheated medium prepared in advance, after mixing well, added dropwise to the above-mentioned feeder cells-containing 96-well plate, 100 μl/well, placed in a 37℃5% CO2 incubator for culturing, half-changed after 3-4d, and after 10d, cultured with HT medium again, and after 2 weeks, cultured with 1640 medium containing 10% FBS.
9. During the period, the cloning growth condition in the 96-well plate is observed every day, and the detection of the specific antibody can be started when the hybridoma cells are distributed to 1/10 area of the bottom of the well, so that the needed hybridoma cell line is screened out. Cells with specific secreted antibodies should be cloned in time and frozen. Typically 3-5 subclones are required to obtain cells of stable genotype and stable secretory phenotype, and after a period of incubation a subclone is required again.
Example 2 sequencing of the heavy and light chain variable regions of the B7-H3 antibody
1. Extracting cDNA of hybridoma cells: extracting RNA from the hybridoma cell strain, and reversely transcribing the obtained RNA into cDNA by using an RT-PCR technology; PCR cloning the hybridoma cell heavy chain variable region (mVH) and light chain variable region (mVL) using specifically designed upstream and downstream primers;
mVH and mVL are respectively connected with a cloning vector (pJET cloning vector), the connection product is used for transforming competent bacteria DH5a, and the pJET vector carries an ampicillin (amp+) resistance gene, so that the transformed bacteria can be smeared on an Amp-resistant LB solid medium and cultured overnight at 37 ℃;
3. the bacteria to be coated grow into scattered colonies, and the colonies with clear edges and good growth are selected for further sequencing and identification;
4. according to the sequencing result, the candidate heavy and light chain variable region sequences are reserved, the heavy and light chain variable region sequences matched with the expression vector are amplified again through PCR, the PCR product is connected with a linear expression vector which is pre-treated through double enzyme digestion, the connection product is used for transforming competent bacteria DH5a, and the expression vector is provided with kanamycin (Kana+) resistance genes, so that the transformation bacterial liquid can be coated on Kana resistance LB solid culture medium, and is cultured overnight at 37 degrees;
5. comparing the sequencing results of the two times, selecting the transformant with the correct sequence, and extracting the plasmid after the amplification culture;
6. eukaryotic expression cell strain 293 is cotransfected with an expression vector linked to the desired monoclonal antibody heavy and light chain variable region genes. 293 cells were cultured in suspension, and SFM4Transfx-293 witout L-glutamine (liquid) serum-free medium was passaged and expanded, and replaced with Gibco free ™ 293 Expression Medium serum-free medium at the time of transfection. Harvesting the supernatant after 7 days of continuous culture, centrifuging for 30min at 4000g, removing impurities such as cells in the supernatant, and filtering and sterilizing by using a 0.45um filter;
7. the obtained supernatant contains the target antibody, and the flow detection expression monoclonal antibody is well combined with breast cancer cells M435.
The B7-H3 antibody heavy chain variable region obtained by the above method:
EVQLVESGGGLVKPGGSLKLSCAASGFTFSRYAMSWVRQTPEKRLEWVATISDDGRNTYDRDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCVRHRAITTARFDYWGQGTSLTVSS
three hypervariable regions: GFTFSRYA, ISDDGRNT, VRHRAITTARFDY.
Antibody light chain variable region:
DILMTQSPSSMSVSLGDTVSITCHASQDISSNIGWLQQGPGNSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISGLESEDFADYYTLQSVQFPYTFGGGTKLEIK
three hypervariable regions: QDISSN, HGT, LQSVQFPYT.
The invention extracts a heavy chain variable region (mVH) and a light chain variable region (mVL) from hybridoma cells secreting anti-human B7-H3 monoclonal antibodies, reserves candidate heavy and light chain variable region sequences according to a sequencing result, PCR (polymerase chain reaction) amplifies heavy and light chain variable region sequences matched with an expression vector, connects a PCR product with a linear expression vector which is pre-treated by double enzyme digestion, and converts a connection product into competent bacteria DH5a. Cotransfection of eukaryotic expression cell strain 293 with expression vector connected with target monoclonal antibody heavy chain and light chain variable region gene, culture of the obtained supernatant containing target antibody, and verification of correct sequence of the obtained heavy chain and light chain variable region;
EXAMPLE 3 immunohistochemical techniques
1. Paraffin section
Fresh tissue was taken, trimmed to 1cm×1cm×0.3cm size tissue pieces, fixed in neutral formalin for 24h, dehydrated in a gradient alcohol solution (70% alcohol 15min, 80% alcohol 15min, 90% alcohol 1h, 95% alcohol 2h, absolute alcohol 2 h), xylene-clear (30 min each for two times), waxed (1 h for the first time, 2h for the second time), paraffin-embedded and cut into 5 μm sheets, which were attached to a polylysine-coated slide.
2. Immunohistochemical staining
0.03% H 2 O 2 Methanol incubation for 30min, 10% BSA dropwise, room temperature incubation for 30min, no washing, primary antibody dropwise (dilution of anti-human CD105 antibody is 1:100), PBS instead of primary antibody as negative control, incubation at 37 ℃ for 2H, PBS flushing, general secondary antibody dropwise use liquid, incubation at 37 ℃ for 30min, PBS flushing for three times, DAB-H2O2 color development, hematoxylin counterstaining, 1% hydrochloric acid-alcohol differentiation, gradient ethanol dehydration (75% ethanol 3min,95% ethanol I3 min,95% ethanol II 3min,100% ethanol I3 min,100% ethanol II 3 min), air blower blow drying of absolute ethanol, neutral resin sealing, microscopic observation and photographing.
Sequence listing
<110> Asahi optical Star Biotechnology Co., ltd
<120> preparation method of anti-human B7-H3 monoclonal antibody, immunohistochemical detection method and application thereof, and kit thereof
<130> 20180107
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 120
<212> PRT
<213> Artificial sequence
<400> 1
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Asp Asp Gly Arg Asn Thr Tyr Asp Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Val Arg His Arg Ala Ile Thr Thr Ala Arg Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Leu Thr Val Ser Ser
115 120
<210> 2
<211> 8
<212> PRT
<213> Artificial sequence
<400> 2
Gly Phe Thr Phe Ser Arg Tyr Ala
1 5
<210> 3
<211> 8
<212> PRT
<213> Artificial sequence
<400> 3
Ile Ser Asp Asp Gly Arg Asn Thr
1 5
<210> 4
<211> 13
<212> PRT
<213> Artificial sequence
<400> 4
Val Arg His Arg Ala Ile Thr Thr Ala Arg Phe Asp Tyr
1 5 10
<210> 5
<211> 107
<212> PRT
<213> Artificial sequence
<400> 5
Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu Gly
1 5 10 15
Asp Thr Val Ser Ile Thr Cys His Ala Ser Gln Asp Ile Ser Ser Asn
20 25 30
Ile Gly Trp Leu Gln Gln Gly Pro Gly Asn Ser Phe Lys Gly Leu Ile
35 40 45
Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Gly Leu Glu Ser
65 70 75 80
Glu Asp Phe Ala Asp Tyr Tyr Thr Leu Gln Ser Val Gln Phe Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 6
<211> 6
<212> PRT
<213> Artificial sequence
<400> 6
Gln Asp Ile Ser Ser Asn
1 5
<210> 7
<211> 3
<212> PRT
<213> Artificial sequence
<400> 7
His Gly Thr
1
<210> 8
<211> 9
<212> PRT
<213> Artificial sequence
<400> 8
Leu Gln Ser Val Gln Phe Pro Tyr Thr
1 5
Claims (3)
1. An anti-human B7-H3 monoclonal antibody comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain variable region is identical to SEQ ID No.1, and is specifically as follows:
EVQLVESGGGLVKPGGSLKLSCAASGFTFSRYAMSWVRQTPEKRLEWVATISDDGRNTYDRDS VKGRFTISRDNARNTLYLQMSSLRSEDTALYYCVRHRAITTARFDYWGQGTSLTVSS; the heavy chain variable region has three hypervariable regions: GFTFSRYA, ISDDGRNT, VRHRAITTARFDY;
the amino acid sequence of the light chain variable region is the same as SEQ ID NO.2, and the specific steps are as follows:
DILMTQSPSSMSVSLGDTVSITCHASQDISSNIGWLQQGPGNSFKGLIYHGTNLEDGVPSRFSG SGSGADYSLTISGLESEDFADYYTLQSVQFPYTFGGGTKLEIK; the light chain variable region has three hypervariable regions: QDISSN, HGT, LQSVQFPYT.
2. The use of an anti-human B7-H3 monoclonal antibody according to claim 1, wherein the anti-human B7-H3 monoclonal antibody is used for preparing a kit for immunohistochemical detection of tumor tissue.
3. The kit of claim 1, wherein the kit comprises the anti-human B7-H3 monoclonal antibody.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910058619.0A CN109851673B (en) | 2019-01-22 | 2019-01-22 | Preparation method of anti-human B7-H3 monoclonal antibody, immunohistochemical detection method and application thereof, and kit thereof |
| PCT/CN2019/124254 WO2020151384A1 (en) | 2019-01-22 | 2019-12-10 | Preparation method and immunohistochemical test method for anti-human b7-h3 monoclonal antibody, and application and kit of anti-human b7-h3 monoclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910058619.0A CN109851673B (en) | 2019-01-22 | 2019-01-22 | Preparation method of anti-human B7-H3 monoclonal antibody, immunohistochemical detection method and application thereof, and kit thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109851673A CN109851673A (en) | 2019-06-07 |
| CN109851673B true CN109851673B (en) | 2023-07-25 |
Family
ID=66895524
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910058619.0A Active CN109851673B (en) | 2019-01-22 | 2019-01-22 | Preparation method of anti-human B7-H3 monoclonal antibody, immunohistochemical detection method and application thereof, and kit thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN109851673B (en) |
| WO (1) | WO2020151384A1 (en) |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109851673B (en) * | 2019-01-22 | 2023-07-25 | 苏州旭光科星抗体生物科技有限公司 | Preparation method of anti-human B7-H3 monoclonal antibody, immunohistochemical detection method and application thereof, and kit thereof |
| CA3203257A1 (en) * | 2020-12-23 | 2022-06-30 | Li Li | Anti-b7-h3 antibody and uses thereof |
| CN112684185B (en) * | 2020-12-25 | 2024-01-12 | 苏州旭光科星抗体生物科技有限公司 | Soluble B7-H4 quantitative detection kit and application thereof |
| US20240108745A1 (en) | 2021-02-09 | 2024-04-04 | Medilink Therapeutics (Suzhou) Co., Ltd. | Bioactive substance conjugate, preparation method therefor and use thereof |
| WO2022200525A1 (en) | 2021-03-26 | 2022-09-29 | Innate Pharma | Multispecific proteins comprising an nkp46-binding site, a cancer antgienge binding site fused to a cytokine for nk cell engaging |
| WO2022258691A1 (en) | 2021-06-09 | 2022-12-15 | Innate Pharma | Multispecific proteins binding to nkg2d, a cytokine receptor, a tumour antigen and cd16a |
| BR112023025331A2 (en) | 2021-06-09 | 2024-02-27 | Innate Pharma | MULTI-SPECIFIC PROTEIN, PHARMACEUTICAL COMPOSITION, RECOMBINANT CELL, NUCLEIC ACID OR NUCLEIC ACID SET, USE OF A PROTEIN OR COMPOSITION, METHODS OR USE |
| WO2022258678A1 (en) | 2021-06-09 | 2022-12-15 | Innate Pharma | Multispecific proteins binding to nkp30, a cytokine receptor, a tumour antigen and cd16a |
| WO2023272924A1 (en) * | 2021-06-30 | 2023-01-05 | 徐州医科大学 | Novel fully human antibody for human b7h3, chimeric antigen receptor and uses thereof |
| CN113683697B (en) * | 2021-08-27 | 2022-06-17 | 上海祥耀生物科技有限责任公司 | anti-B7-H3 antibody, preparation method and application thereof |
| CN116751297A (en) * | 2021-12-15 | 2023-09-15 | 苏州旭光科星抗体生物科技有限公司 | ELISA detection kit for detecting content of soluble B7-H5 protein |
| CN114380910B (en) * | 2022-01-07 | 2023-04-28 | 苏州旭光科星抗体生物科技有限公司 | Humanized monoclonal antibody targeting human B7-H3 molecule and application thereof |
| CN118647408A (en) | 2022-02-16 | 2024-09-13 | 苏州宜联生物医药有限公司 | Conjugate of antibody-eribulin or derivative thereof, intermediate, preparation method, pharmaceutical composition and application |
| CN114773477B (en) * | 2022-06-23 | 2022-09-09 | 上海优替济生生物医药有限公司 | anti-B7-H3 antibodies and uses thereof |
| CN117362434A (en) * | 2022-06-30 | 2024-01-09 | 南京北恒生物科技有限公司 | Antibodies targeting B7-H3 and uses thereof |
| CN116041518A (en) * | 2022-12-26 | 2023-05-02 | 北京清辉联诺生物科技有限责任公司 | Antibodies or antibody fragments targeting B7-H3, and their use in the field of chimeric antigen receptor immune cell therapies |
| CN116514975B (en) * | 2023-03-14 | 2024-02-09 | 苏州旭光科星抗体生物科技有限公司 | Mouse anti-human B7-H3 monoclonal antibody, preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101104639A (en) * | 2006-07-10 | 2008-01-16 | 苏州大学 | Preparation for anti human B7-H3 monoclonal antibody and application thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2011223782B2 (en) * | 2010-03-04 | 2014-09-18 | Macrogenics, Inc. | Antibodies reactive with B7-H3, immunologically active fragments thereof and uses thereof |
| US8802091B2 (en) * | 2010-03-04 | 2014-08-12 | Macrogenics, Inc. | Antibodies reactive with B7-H3 and uses thereof |
| TWI561531B (en) * | 2011-04-25 | 2016-12-11 | Daiichi Sankyo Co Ltd | Anti b7-h3 antibody |
| JP6636016B2 (en) * | 2014-05-29 | 2020-01-29 | スプリング バイオサイエンス コーポレーション | Anti-B7-H3 antibody and diagnostic use thereof |
| WO2018161872A1 (en) * | 2017-03-06 | 2018-09-13 | 江苏恒瑞医药股份有限公司 | Anti-b7-h3 antibody, antigen-binding fragment thereof, and pharmaceutical use thereof |
| CN109851673B (en) * | 2019-01-22 | 2023-07-25 | 苏州旭光科星抗体生物科技有限公司 | Preparation method of anti-human B7-H3 monoclonal antibody, immunohistochemical detection method and application thereof, and kit thereof |
-
2019
- 2019-01-22 CN CN201910058619.0A patent/CN109851673B/en active Active
- 2019-12-10 WO PCT/CN2019/124254 patent/WO2020151384A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101104639A (en) * | 2006-07-10 | 2008-01-16 | 苏州大学 | Preparation for anti human B7-H3 monoclonal antibody and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2020151384A1 (en) | 2020-07-30 |
| CN109851673A (en) | 2019-06-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109851673B (en) | Preparation method of anti-human B7-H3 monoclonal antibody, immunohistochemical detection method and application thereof, and kit thereof | |
| CN108948204B (en) | Anti-human B7-H4 monoclonal antibody, identification method, application and kit | |
| CN110461881B (en) | Chimeric antigen receptor | |
| CN112661842B (en) | anti-Ki-67 specific monoclonal antibody and application thereof | |
| CN112500484B (en) | anti-TROP 2 antibody and application thereof | |
| CN108084263B (en) | Anti-human CD25 chimeric monoclonal antibody and preparation method and application thereof | |
| CN111995683B (en) | anti-PD-L1 protein monoclonal antibody, cell strain, preparation method and application thereof | |
| CN116514975B (en) | Mouse anti-human B7-H3 monoclonal antibody, preparation method and application thereof | |
| WO2019085238A1 (en) | Preparation method of anti-human b7-h1 monoclonal antibody and application of same in immunohistochemical detection | |
| CN111187351B (en) | Liver cancer detection kit | |
| JP2000506379A (en) | Cell lines and clones of human prostate epithelial immortal cells and their research and application for prostate cancer treatment | |
| Zhang et al. | The study of the tumor stem cell properties of CD133+ CD44+ cells in the human lung adenocarcinoma cell line A549 | |
| CN114316048B (en) | Enzyme-linked immunoassay kit for detecting content of soluble B7-H5 protein and application thereof | |
| CN107365383A (en) | A kind of monoclonal antibodies of Humanized anti-human PD 1 and preparation method thereof, purposes and medicine | |
| CN109053889B (en) | A kind of anti-human PD1 monoclonal antibody and purposes | |
| CN108948202A (en) | A kind of anti-human PD1 monoclonal antibody and application thereof | |
| WO2008093886A1 (en) | Mcf7-derived cell | |
| CN103352053A (en) | Exogenous-gene removable slow virus controlled expression carrier system and application | |
| CN112430570B (en) | Use of adipose-derived stem cells edited by gene editing technology for improving differentiation efficiency | |
| CN110106182B (en) | Application of p65 gene in porcine ovarian granulosa cells | |
| CN116217726A (en) | Anti-human HHA 2 monoclonal antibody, preparation and application thereof | |
| CN102827813B (en) | Anti-human CD133 monoclonal antibody and preparation method and applications thereof | |
| CN116478285B (en) | Diagnostic mouse-derived anti-human CK antibody and preparation method and application thereof | |
| CN110218725B (en) | Long-chain non-coding RNA and application thereof | |
| CN117025547B (en) | Hybridoma cell strain for producing anti-B7H 3 monoclonal antibody and application thereof |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20191127 Address after: 215000 Room 302, building 12, No. 8, Jinfeng Road, Suzhou high tech Zone, Suzhou, Jiangsu Province Applicant after: Suzhou Xuguang Kexing antibody Biotechnology Co.,Ltd. Address before: 215063 303, 305, 15, 8 Jinfeng Road, Suzhou high tech Zone, Jiangsu Applicant before: SUZHOU BRIGHT SCISTAR BIOTECHNOLOGY Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |