CN108948204B - Anti-human B7-H4 monoclonal antibody, identification method, application and kit - Google Patents
Anti-human B7-H4 monoclonal antibody, identification method, application and kit Download PDFInfo
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- 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
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- 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
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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- 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
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- 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
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- 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
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- 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
- G01N2333/70532—B7 molecules, e.g. CD80, CD86
Abstract
The invention discloses an anti-human B7-H4 monoclonal antibody, an identification method, application and a kit, wherein the anti-human B7-H4 monoclonal antibody and the monoclonal antibody can be used for immunohistochemical specimen detection and tumor tissue B7-H4 expression analysis, a heavy chain variable region (mVH) and a light chain variable region (mVL) are extracted from a hybridoma cell secreting the anti-human B7-H4 monoclonal antibody, and a B7-H4 heavy chain and light chain variable sequence is verified by sequencing. On the basis, the eukaryotic cell strain is implemented to express the antibody, the obtained antibody has good binding capacity after verification, the tumor specimen is specifically dyed by adopting immunohistochemical detection, and the dyeing effect and specificity of the self-developed B7-H4 monoclonal antibody and the commercialized B7-H4 antibody on the same tissue specimen are compared.
Description
Technical Field
The invention relates to detection application of antibody immunohistochemistry and verification of sequences of heavy chain variable regions and light chain variable regions of antibodies, belongs to the technical field of biology, and particularly relates to an anti-human B7-H4 monoclonal antibody, an identification method, application and a kit.
Background
In recent years, more and more researches show that the B7-H4 molecule is abnormally expressed in various tumor tissues, such as ovarian cancer, breast cancer, lung cancer, renal cancer, colorectal cancer, prostate cancer and the like. Clinical review of pathological cases revealed that B7-H4 expression correlated with clinical pathology, survival analysis revealed poor prognosis in cases with high B7-H4 expression, high mortality, and high levels of soluble B7-H4 (sB 7-H4) in peripheral blood of tumor patients. In addition, B7-H4 is relevant to graft immune rejection and autoimmune pathology, and the graft transfected with the B7-H4 gene can inhibit the activity of host T cells, so that the survival time of the graft is prolonged. The research results all indicate that B7-H4 is an important negative co-stimulatory molecule and has important research and application values.
In many tumors in humans, B7-H4 is expressed at both the mRNA and protein levels and is inversely correlated with patient prognosis. The B7-H4 is abnormally expressed in tumor tissues or peripheral blood of tumor patients and is closely related to age, pathological type, tumor biological effect and survival rate of the patients, so that the development of the mouse anti-human B7-H4 monoclonal antibody can lay a material foundation for further researching the action of the molecule in the tumor, and simultaneously provides an effective research means for exploring a B7-H4 signal conduction path and mechanism, and the functional anti-human B7-H4 monoclonal antibody is more expected to become a potential target spot for tumor biological treatment.
Disclosure of Invention
One of the purposes of the present invention is to provide an anti-human B7-H4 monoclonal antibody;
the second purpose of the invention is to provide a method for identifying the variable region sequences of the heavy chain and the light chain of the anti-human B7-H4 antibody. The invention extracts heavy chain variable region (mVH) and light chain variable region (mVL) from hybridoma cells secreting anti-human B7-H4 monoclonal antibody, reserves candidate heavy chain and light chain variable region sequences according to sequencing results, amplifies heavy chain and light chain variable region sequences matched with an expression vector by PCR, connects PCR products with a linear expression vector pretreated by double enzyme digestion, and converts the connected products into competent bacteria DH5a. The expression vector connected with the genes of the heavy chain and the light chain variable regions of the target monoclonal antibody is cotransfected with the eukaryotic expression cell strain 293, and the supernatant obtained by culturing contains the target antibody, thereby proving that the sequences of the obtained heavy chain and light chain variable regions are correct;
the invention also aims to provide an anti-human B7-H4 monoclonal antibody for immunohistochemical detection of a tissue specimen, and the antibody B7-H4 is found to carry out histochemical staining on a liver cancer tissue specimen tissue, so that a cell membrane and a cytoplasm are obviously colored; the kit is further used for detecting a pathological specimen of immunohistochemistry, and the result shows that B7-H4 is highly expressed in cytoplasm of interstitial cells of intestinal cancer patients, cell nucleuses of intestinal cancer patients and cell membranes of intestinal cancer cells.
The fourth object of the present invention is to provide a kit prepared using the above-described anti-human B7-H4 monoclonal antibody.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the anti-human B7-H4 monoclonal antibody as the first aspect of the invention comprises a heavy chain and a light chain, and is characterized in that the amino acid sequence of a heavy chain variable region (mVH) is the same as that of SEQ ID NO.1, and specifically comprises the following steps:
DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLDWMGYISYSGDTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARDYGSGAWYFDVWGAGTTVTVSS；
the amino acid sequence of the light chain variable region (mVL) is the same as that of SEQ ID NO.2, and the amino acid sequence is as follows:
DIVMTQSPSSLAVSVGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFILTISSVQAEDLAVYYCKQSYNLWTFGGGTKLEIK。
in a preferred embodiment of the invention, the heavy chain variable region (mVH) has three hypervariable regions: gysitsdfa, ISYSTDT, ARDDYYGSGAWYFDV.
In a preferred embodiment of the invention, the light chain variable region (mVL) has three hypervariable regions: QSLLNSRTRKNY, WAS, KQSYNLWT.
The method for identifying sequences of variable regions of heavy and light chains of an anti-human B7-H4 antibody as a second aspect of the present invention comprises the steps of:
(1) Preparing a hybridoma of the B7-H4 monoclonal antibody;
(1.1) immunizing a mouse;
(1.2) cell culture;
(1.3) fusion and screening;
(2) B7-H4 mouse/human chimeric antibody preparation;
(2.1) extracting cDNA of hybridoma cells: extracting RNA from the hybridoma cell strain, and performing reverse transcription on 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 a PCR method through a specially designed upstream primer and a specially designed downstream primer; the sequences of the specially designed upstream and downstream primers are respectively nucleotide SEQ ID NO.3 and nucleotide sequence 4;
(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 transformed into a competent bacterium DH5a, and as the pJET vector carries an ampicillin (Amp +) resistance gene, a transformation bacterium solution can be coated on an Amp-resistant LB solid culture medium and cultured at 37 ℃ overnight;
(2.3) the bacteria to be plated grow dispersed colonies, selecting the colonies with clear edges and good growth, and further sequencing and identifying;
(2.4) reserving candidate sequences of a heavy chain variable region (mVH) and a light chain variable region (mVL) according to a sequencing result, performing PCR amplification again to obtain sequences of the heavy chain variable region (mVH) and the light chain variable region (mVL) matched with an expression vector, connecting a PCR product with a double-enzyme digestion pre-treated linear expression vector, connecting a product to transform competent bacteria DH5a, and coating a transformed bacterium liquid on a Kana-resistant LB solid medium due to the fact that the expression vector has a kanamycin (Kana +) resistance gene, and performing overnight culture at 37 ℃;
(2.5) comparing the two sequencing results according to the step (2.4) in the sequencing method, selecting a transforming strain with a correct sequence, and performing plasmid extraction after amplification culture;
(2.6) cotransfecting the eukaryotic expression cell strain 293 with an expression vector connected with a target single-antibody heavy chain variable region (mVH) gene and a target light chain variable region (mVL) gene;
(2.7) the obtained supernatant contains the target antibody, the monoclonal antibody expressed by flow detection is well combined with M435, and the positive rate reaches more than 95%, which indicates that the correct sequences of the heavy chain and light chain variable regions of the antibody 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 with L-glutamine (liquid) serum-free Medium is subcultured, and replaced with Gibco FreeStyle 293 Expression Medium serum-free Medium during transfection; after continuous culture for 7 days, the supernatant was harvested, centrifuged at 4000g for 30min to remove impurities such as cells from the supernatant, and sterilized by filtration through a 0.45um filter.
The application of the anti-human B7-H4 monoclonal antibody as the third aspect of the invention is characterized in that the anti-human B7-H4 monoclonal antibody is used for preparing a kit for immunohistochemical detection of tumor tissues.
The kit according to the fourth aspect of the present invention is characterized by containing the above-described anti-human B7-H4 monoclonal antibody.
The invention has the beneficial effects that:
the anti-human B7-H4 monoclonal antibody provided by the invention can be used for immunohistochemical detection of tumor tissues, improves the precision of 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, particularly low-differentiation or undifferentiated tumors.
Drawings
FIG. 1 is a schematic diagram showing the recognition of B7-H4 protein by anti-human B7-H4 monoclonal antibody 4B2 in Western blot analysis, and the results show that monoclonal antibody 4B2 can specifically recognize and bind to B7-H4 protein in cell lysate, the target band is clear, and negative control Isotype and CHO/mock groups have no binding band. In the figure: igG Isotype, isotype antibody negative control; 4B2 is the anti-human B7-H4 monoclonal antibody of the invention; CHO/mock cell lysate, CHO/B7-H4 cell lysate.
FIG. 2 is a schematic diagram showing the karyotype analysis (1000-fold) of the obtained hybridoma by using the nuclear chromosome technique, and the results of the karyotype analysis of the hybridoma cells in the figure show that the number of chromosomes of the hybridoma cell lines is more than 80, and exceeds the number of chromosomes of mouse B cells and SP2/0 cells, indicating that the hybridoma cell lines are fused cells.
FIG. 3 is a schematic diagram of anti-human B7-H4 antibody used for immunohistochemical detection of liver cancer tissue, in which 4B2 immunohistochemical detection of liver cancer tissue specimen, CST is positive control; the results show that B7-H4 is highly expressed in liver cancer tissues, and both CST and 4B2 antibodies stain the tissues.
FIG. 4 is a schematic diagram of a clinical colon cancer patient specimen detected by anti-human B7-H4 antibody in immunohistochemical detection: the figure shows that the anti-human B7-H4 molecule is not basically expressed in benign lesion stage, is dispersedly expressed in the colorectal inflammation tissue, gradually increases the expression adenoma stage, and is highly expressed in the colorectal cancer tissue.
Detailed Description
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
Unless otherwise specified, the examples are all routine experimental techniques in the art.
The sources of biomaterial used in the examples are as follows:
EXAMPLE 1 obtaining of hybridoma producing anti-human B7-H4 monoclonal antibody
(I) immunizing mice
The fusion protein or the transgenic cell is used for immunizing a mouse for four times, the interval is 21 days every time, the orbital blood titer of the mouse is measured after the fourth immunization for 7-10 times, and the boosting immunization is carried out if the titer is measured well.
(II) cell culture
1.1 mouse of BALB/c 6 to 7 weeks old is taken and placed in 75% ethanol solution for 2min 1d before fusion.
2. The spleens of the mice were aseptically removed, placed in a 200 mesh stainless steel screen, and ground to obtain a single cell suspension. Wash twice (1400rpm, 5min) with 1640 basic medium for use. Adjusting the cell concentration to 2X 10% with 15% of the Medium 1640 of FBS 5 Per ml, was added dropwise to a 96-well plate at a concentration of 100. Mu.L/well, 37 ℃ and 5% CO 2 Culturing in an incubator.
3. The culture was overnight and observed under low power microscope the next day. And 150ul of subcloned cells were plated.
(III) fusion and screening
Preparing:
1. equipment: a mouse soaking cup; a simple dissecting table; a hybridoma package; a heat-preservation water bath cup; a thermometer; 96-well culture plates.
2. Reagent: 75% ethanol solution; preheating 1640 basic culture medium at 37 ℃; preheating 1ml of PEG at 37 ℃; preheating 1640 basic culture medium 14ml (PEG stop solution) at 37 ℃; medium was selected by pre-heating 1640 at 37 ℃ (15% FBS is contained, and the amount of HAT added was calculated so that the final concentration of HAT in the medium in the final 96-well plate was 1%).
The method comprises the following steps:
1. the immunized mice were washed with running water and placed in 75% ethanol solution for 2min.
2. The spleens of the mice were aseptically removed, placed in a 200 mesh stainless steel screen, and ground to obtain a single cell suspension. Washed twice (1400rpm, 5min) with preheated 1640 basic medium for use.
3. SP2/0 cells that grew well and were in the logarithmic growth phase were collected and washed twice (1400 rpm, 5min) with pre-heated 1640 basic medium for future use.
4. SP2/0 cells or Ag8 cells were mixed with spleen cells in a 50ml clear plastic centrifuge tube, the ratio of spleen cells to myeloma cells was typically 5, the cells were washed once (1400 rpm,5 min) with pre-heated 1640 basic medium, the supernatant was discarded (to avoid unnecessary dilution of PEG) and the tube bottom was rubbed with the palm (or flicked with the fingers) to mix the two cells well into suspension cells.
5. Placing the centrifuge tube in a 37 ℃ heat-preservation water bath cup for preheating, sucking 1ml of 50% PEG solution preheated at 37 ℃, completing adding at a constant speed within 1min, slightly shaking the centrifuge tube while adding, and slightly shaking in the 37 ℃ water bath for 60s after adding. (one drop for 3 seconds)
6. Add gently 14ml 1640 basal medium pre-warmed at 37 ℃ along the walls of the tubes (1 ml for 1min, 3ml for 3min and finally 10ml slowly). (Uniform dropping)
After standing at 7.37 ℃ for 5min, the mixture was centrifuged (800rpm, 5 min), and the supernatant was discarded (the tube was tilted, and the supernatant was aspirated).
8. The precipitated cells were gently resuspended (not blown) in a 1640 selective medium preheated at 37 ℃ and added to a preheated medium prepared in advance, and then the mixture was dropped into the above 96-well feeder cell-containing culture plate at 100 μ l/well and cultured in a 5% CO2 incubator at 37 ℃ after mixing, half of the culture was changed after 3-4d, and after 10d, the cells were cultured in HT medium, and after 2 weeks, the cells were cultured in the 1640 medium containing 10 FBS.
9. During the period, the clone growth condition in a 96-well plate is observed every day, and generally when hybridoma cells are distributed on 1/10 area of the bottom of a hole, the specific antibody can be detected, and the required hybridoma cell line can be screened. For cells with specific secretion antibodies, the cells should be cloned and frozen in time. It usually takes 3-5 subclones to obtain cells of stable genotype and stable secretory phenotype, and after a period of culture it needs to be subcloned again.
Example 2 sequencing of heavy and light chain variable regions of anti-human B7-H4 antibodies
1. Extraction of cDNA from hybridoma cells: extracting RNA from the hybridoma cell strain, and performing reverse transcription on the obtained RNA into cDNA by using an RT-PCR technology; cloning heavy chain variable region (mVH) and light chain variable region (mVL) of the hybridoma cell by using an upstream primer and a downstream primer which are specially designed;
2. mVH and mVL are respectively connected with a cloning vector (pJET cloning vector), the connection product is transformed into a competent bacterium DH5a, and the pJET vector carries an ampicillin (Amp +) resistance gene, so that a transformation bacterium solution can be coated on an Amp-resistant LB solid culture medium and cultured at 37 ℃ overnight;
3. the method comprises the following steps of (1) growing dispersed colonies by bacteria to be plated, selecting colonies with clear edges and good growth, and further sequencing and identifying;
4. reserving a candidate heavy-light chain variable region sequence according to a sequencing result, performing PCR amplification again to obtain a heavy chain variable region sequence and a light chain variable region sequence which are matched with an expression vector, connecting a PCR product with a linear expression vector subjected to double digestion pretreatment, connecting a product to transform competent bacteria DH5a, coating a transformed bacterium liquid on a Kana resistant LB solid medium due to the fact that the expression vector has a kanamycin (Kana +) resistance gene, and performing overnight culture at 37 ℃;
5. comparing the two sequencing results according to the sequencing method 4, selecting the transformation bacteria with the correct sequence, carrying out amplification culture, and then carrying out plasmid extraction;
6. the expression vector connected with the target monoclonal antibody heavy chain and light chain variable region genes cotransfects the eukaryotic expression cell strain 293. 293 cells were suspension cultured, SFM4Transfx-293 with L-glutamine (liquid) serum-free Medium was passage-expanded, and replaced with Gibco FreeStyle 293 Expression Medium serum-free Medium during transfection. Harvesting 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 harvested supernatant contains the target antibody, and the expressed monoclonal antibody is well combined with the transgenic cell CHO/B7-H4 by Western blot detection.
The heavy chain variable region of the anti-human B7-H4 antibody obtained by the method comprises the following steps:
DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLDWMGYISYSGDTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARDYGSGAWYFDVWGAGTTVTVSS
three hypervariable regions: gysitsdfa, ISYSTDT, ARDDYYGSGAWYFDV.
Antibody light chain variable region:
DIVMTQSPSSLAVSVGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFILTISSVQAEDLAVYYCKQSYNLWTFGGGTKLEIK
three hypervariable regions: QSLLNSRTRKNY, WAS, KQSYNLWT.
Example 3 analysis of chromosome karyotype of hybridoma cells
The colchicine is added into the well-grown cells to make the final concentration of the colchicine be 0.04 to 0.08 mul/ml. Culturing for 2h, collecting cells (1000rpm, 10min) in a centrifuge tube, dropwise adding 0.5ml of 0.075mol/L KCl solution pre-warmed at 37 ℃, then supplementing 5 to 10ml, lightly blowing and beating uniformly by using a suction tube, and incubating for 20min at 37 ℃. 1ml of freshly prepared fixative (3 parts methanol plus 1 part glacial acetic acid, prepared just before use) was added to the tube, centrifuged at 1000rpm for 10min and the supernatant discarded. Then adding 8-10ml of fixing solution, blowing and beating uniformly by using a suction pipe, fixing for 15-20min, centrifuging, and removing supernatant. Then 5ml of the fixing solution is added, and the fixing is carried out for 30min. Centrifuging, removing supernatant, adding 1.5ml of stationary liquid, and blowing and beating uniformly. And (3) taking a glass slide frozen at the temperature of minus 10 ℃, dropwise adding 1 to 2 drops of cell suspension, dyeing for 10 to 20min by using a fresh Giemsa solution (1 part of Giemsa stock solution is added with 9 parts of 0.075mol/L phosphate buffer solution with the pH value of 6.8), washing by running water, and airing. Transparent xylene for three times, and sealing with neutral resin. The specimen with good chromosome dispersion, no overlapping and no chromosome dispersion is selected under a microscope, observed and recorded under an oil microscope and subjected to photomicrography.
Example 4 immunohistochemical techniques
1. Paraffin section
Taking fresh tissues, trimming the tissues into tissue blocks with the size of 1cm multiplied by 0.3cm, fixing the tissues in neutral formalin solution for 24 hours, dehydrating the tissues by gradient alcohol solution (70% alcohol for 15min, 80% alcohol for 15min, 90% alcohol for 1 hour, 95% alcohol for 2 hours and absolute ethyl alcohol for 2 hours), transparent xylene (for two times and 30 minutes for each time), soaking wax (for two times, for 1 hour and 2 hours for the second time), cutting 5 mu m slices after paraffin embedding, and attaching the slices to a polylysine-coated slide glass.
2. Immunohistochemical staining
0.03%H 2 O 2 Methanol incubation for 30min, dropwise addition of 10% BSA, incubation at room temperature for 30min, do notWashing, dropwise adding primary antibody (the dilution of an anti-human CD105 antibody is 1.
Sequence listing
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<120> anti-human B7-H4 monoclonal antibody, identification method, application and kit
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Claims (3)
1. An anti-human B7-H4 monoclonal antibody comprises a heavy chain and a light chain, and is characterized in that the amino acid sequence of a heavy chain variable region is the same as that of SEQ ID NO.1, and the specific amino acid sequence is as follows: <xnotran> DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLDWMGYISYSGDTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARDYGSGAWYFDVWGAGTTVTVSS; </xnotran>
The amino acid sequence of the light chain variable region is the same as that of SEQ ID NO.2, and the specific amino acid sequence is as follows: <xnotran> DIVMTQSPSSLAVSVGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFILTISSVQAEDLAVYYCKQSYNLWTFGGGTKLEIK; </xnotran>
The heavy chain variable region has three hypervariable regions: gysitsdfa, ISYSTDT, ARDYGSGAWYFDV; the light chain variable region has three hypervariable regions: QSLLNSRTRKNY, WAS, KQSYNLWT.
2. The use of the monoclonal antibody against human B7-H4 as claimed in claim 1, wherein the monoclonal antibody against human B7-H4 is used for preparing a kit for immunohistochemical detection of tumor tissues.
3. A kit comprising an anti-human B7-H4 monoclonal antibody according to claim 1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810619746.9A CN108948204B (en) | 2018-06-15 | 2018-06-15 | Anti-human B7-H4 monoclonal antibody, identification method, application and kit |
| PCT/CN2018/094692 WO2019237442A1 (en) | 2018-06-15 | 2018-07-05 | Anti-human b7-h4 monoclonal antibody, identification method, use and kit |
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| CN201810619746.9A CN108948204B (en) | 2018-06-15 | 2018-06-15 | Anti-human B7-H4 monoclonal antibody, identification method, application and kit |
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| CN108948204A CN108948204A (en) | 2018-12-07 |
| CN108948204B true CN108948204B (en) | 2023-01-13 |
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| CN109988752A (en) * | 2019-03-25 | 2019-07-09 | 上海百融济生物科技有限公司 | A kind of anti-human B7-H4 monoclonal antibody and its preparation and application |
| 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 |
| JP2024501894A (en) | 2021-01-04 | 2024-01-16 | メルサナ セラピューティクス インコーポレイテッド | B7H4 targeting antibody-drug conjugates and methods of use thereof |
| CN113156120B (en) * | 2021-03-26 | 2022-03-25 | 中国医学科学院北京协和医院 | Application of B7H4 in preparation of endometrial cancer molecular typing reagent and system |
| CN117147882B (en) * | 2023-10-31 | 2024-01-26 | 中国农业科学院生物技术研究所 | Kit and method for quantitative detection of insect-resistant protein Cry1Ah enzyme-linked immunity |
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| WO2014159835A1 (en) * | 2013-03-14 | 2014-10-02 | Genentech, Inc. | Anti-b7-h4 antibodies and immunoconjugates |
| CN103981150B (en) * | 2014-03-28 | 2016-06-01 | 苏州大学 | Anti-human B7-H4 monoclonal antibody and Synthesis and applications thereof |
| JP6994223B2 (en) * | 2016-07-26 | 2022-02-04 | 静岡県 | Anti-B7-H4 antibody |
| CN108084263B (en) * | 2016-12-16 | 2021-07-13 | 苏州旭光科星抗体生物科技有限公司 | Anti-human CD25 chimeric monoclonal antibody and preparation method and application thereof |
| CN107299085B (en) * | 2017-05-26 | 2020-09-29 | 广东医科大学 | Hybridoma cell strain secreting anti-human B7-H4 extracellular monoclonal antibody, anti-human B7-H4 monoclonal antibody and application of anti-human B7-H4 extracellular monoclonal antibody |
| CN108084264A (en) * | 2017-10-31 | 2018-05-29 | 苏州旭光科星生物技术有限公司 | Anti-human B7-H1 method for preparing monoclonal antibody and its immunohistochemistry detect apply |
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