CN101012280A - Method of preparing monoclonal antibody against human soluble mesothelin related protein - Google Patents
Method of preparing monoclonal antibody against human soluble mesothelin related protein Download PDFInfo
- Publication number
- CN101012280A CN101012280A CN 200710061497 CN200710061497A CN101012280A CN 101012280 A CN101012280 A CN 101012280A CN 200710061497 CN200710061497 CN 200710061497 CN 200710061497 A CN200710061497 A CN 200710061497A CN 101012280 A CN101012280 A CN 101012280A
- Authority
- CN
- China
- Prior art keywords
- cell
- serum
- centrifugal
- related protein
- human soluble
- 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
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a making method of relative protein mono-cloning antibody of anti-human soluble mesothelium, which is characterized by the following: predicting multi-parameter on the antigen surface position of human mesothelium B; making anti-human SMR mono-cloning antibody; making the monomer-cloning antibody (2H10 and 6D9) of relative protein; possessing higher specificity.
Description
Technical field
The invention belongs to the preparation of antibody, be meant a kind of anti-human soluble mesothelin related protein MONOCLONAL ANTIBODIES SPECIFIC FOR method especially.
Background technology
(mesothelin MSLN) is the cell surface glycoprotein that a kind of molecular weight is about 40kD, high expression level in malignant tumours such as mesothelioma, ovarian cancer, carcinoma of the pancreas to the mesothelium element.The plain cDNA coding molecule of mesothelium amount is the precursor protein of 69kD, be the fragment of 40kD and 31kD by protease hydrolysis after the glycosylation, wherein the fragment of 40kD links to each other with after birth by glycosyl-phosphatidyl inositol (glycosylphosphatidylinositol), is called the mesothelium element; After coming off, the fragment of 31kD is called megakaryocyte colony stimulating factor (megakaryocyte-potentiating factor).The mesothelium element comprises two isomer, wherein isomer 2 is owing to the intron that has kept between the 16th and 17 exons, make its protein translation premature termination, come off from cell surface and to form soluble mesothelin related protein (soluble mesothelin relatedproteins, SMR), molecular weight is about 42～45kD.The malignant tumor patient of the plain positive expression of mesothelium can detect the existence of soluble mesothelin related protein in its serum and the ascites, thereby make it to become new malignant tumour monitoring index in the cancerous tissue.But up to the present, still there is not the enzyme linked immunological kit that can detect soluble mesothelin.
Summary of the invention
The polypeptide that the objective of the invention is to synthesize human soluble mesothelin related protein has prepared monoclonal antibody, and its biological specificity is identified.
Overall technical architecture of the present invention is:
Anti-human soluble mesothelin related protein MONOCLONAL ANTIBODIES SPECIFIC FOR method comprises following processing step:
The multi-parameter prediction and the antigen peptide of A, the plain B cell antigen epi-position of people's mesothelium are synthetic
(1) one-parameter is predicted respectively
Use the plain aminoacid sequence total length of GOLDKEY software analysis mesothelium and analyze the plain aminoacid sequence total length of mesothelium, use Hopp ﹠amp; Woods wetting ability parameter, Welling antigenicity parameter, accessibility parameter, secondary structure are carried out the one-parameter prediction respectively to epitope, and the peak that all is higher than thresholding is the site that dopes; Wherein the plain aminoacid sequence total length of mesothelium is as follows:
1 malptarpll?gscgtpalgs?llfllfslgw?vqpsrtlage?tgqeaapldg?vlanppniss
61 lsprqllgfp?caevsglste?rvrelavala?qknvklsteq?lrclahrlse?ppedldalpl
121?dlllflnpda?fsgpqactrf?fsritkanvd?llprgaperq?rllpaalacw?gvrgsllsea
181?dvralgglac?dlpgrfvaes?aevllprlvs?cpgpldqdqq?eaaraalqgg?gppygppstw
241?svstmdalrg?llpvlgqpii?rsipqgivaa?wrqrssrdps?wrqpertilr?prfrrevekt
301?acpsgkkare?ideslifykk?weleacvdaa?llatqmdrvn?aipftyeqld?vlkhkldely
361?pqgypesviq?hlgylflkms?pedirkwnvt?sletlkalle?vnkghemspq?aprrplpqva
421?tlidrfvkgr?gqldkdtldt?ltafypgylc?slspeelssv?ppssiwavrp?qdldtcdprq
481?ldvlypkarl?afqnmngsey?fvkiqsflgg?aptedlkals?qqnvsmdlat?fmklrtdavl
541?pltvaevqkl?lgphveglka?eerhrpvrdw?ilrqrqddld?tlglglqggi?pngylvldls
601?mqealsgtpc?llgpgpvltv?lalllas
Above-mentioned aminoacid sequence is taken passages from NCBI protein sequence storehouse
(2) integrated forecasting
Use the method contrast in antigenicity index analysis curve and the above-mentioned steps (1), to avoid error, the aminoacid sequence of finally having determined antigen site is Q D L D T C D P R Q L;
(3) antigen peptide is synthetic
Utilize tyrosine to have the special construction of two amino, synthesize earlier and have 4 aminoterminal tyrosine cores, peptide chain extends to the N end from the C end, has been to press previously selected aminoacid sequence synthetic (synthetic by the polypeptide lab A BI433A of Xuan Wu, Beijing hospital type polypeptide automatic DNA synthesizer DNA) with each amino;
B, mouse-anti people SMR MONOCLONAL ANTIBODIES SPECIFIC FOR
(1) mouse boosting cell preparation
Nitrocellulose filter is made diaphragm, soaked overnight in containing the antigenic solution of the compound peptide of SMR, the subcutaneous immune BALB/c mouse in first back, after behind the initial immunity 10～14 days, 20～28 days, carry out booster immunization twice, after the last immunity 3～10 days, under aseptic condition, take out spleen, grinding makes splenocyte free, adopt GKN damping fluid washing back centrifugal, sedimentary cell is resuspended with 10～30ml serum-free DMEM nutrient solution, and it is stand-by to put into CO2gas incubator;
(2) myeloma cell's preparation
SP2/0 myeloma cell strain elder generation before fusion does to adapt to the substratum that contains the 8-azaguanine cultivates, and getting the mouse peritoneal cell is feeder cell, and the adjustment cell concn is 1～3 * 10
5/ ml placed culture plate to cultivate in 24～48 hours in advance; Cytogamy same day, collect the SP2/0 cell that is in logarithmic phase, add serum-free DMEM substratum 10～30ml carry out centrifugal, with 10～20ml serum-free DMEM substratum resuspension cell, the mensuration cytoactive;
(3) cytogamy
With myeloma cell and splenocyte by 1: 7～10 mixed, with serum-free medium washing, centrifugal back supernatant discarded, at room temperature splenocyte and SP2/0 myeloma cell are merged, polyoxyethylene glycol 0.5～3ml the effect 0.5～3 minute that adds 37 ℃ again, add serum-free medium 0.5～10ml, end the polyoxyethylene glycol effect, it is centrifugal to add serum-free DMEM 3～10ml, removes supernatant; Add the HAT substratum that contains 20% bovine serum, make cell reach 0.5～10
6/ ml; Add and to contain in the culture plate of feeder cell, in 10～200 μ l/ holes, 37 ℃, 5% carbonic acid gas incubator, cultivate;
(4) detect antibody titer
Detect the positive colony that each hole produces anti-SMR antibody with the indirect enzyme-linked immunosorbent method;
(5) a large amount of preparations of antibody
Adopting the gauge dilution method to carry out mono-clonal to the positive colony cell cultivates, filters out positive clone strain 2H10 and 6D9; The mouse peritoneal injection is adopted in a large amount of preparations of antibody, prior to BALB/c mouse abdominal injection whiteruss, and 2～4 all pneumoretroperitoneum injections 1～10 * 10
6Individual hybridoma, inoculating cell was collected ascites after 10～15 days, and the centrifuging and taking supernatant liquor is frozen.
Technical qualification among the present invention in each processing step are:
In the step in the process B (1), it is 0.2～0.5cm * 0.5～1.0cm that nitrocellulose filter is made the diaphragm-operated specification.
In the step in the process B (1), the subcutaneous immune BALB/c mouse in first back adopts entrapping method.
In the step in the process B (1), centrifugal condition is 1000～3000 rev/mins, 5～10 minutes time, abandons supernatant, and repeated centrifugation once.
In the step in the process B (1), the method for booster immunization is identical with the method for initial immunity.
In the step in the process B (2), culture plate is 96 holes, and centrifugal condition is 1000～3000 rev/mins, 5～10 minutes time, 3 times; The method of measuring cytoactive is trypan blue dyeing.
In the step in the process B (3), be 500～3000 rev/mins with serum-free medium washing, centrifugal condition in centrifugal, 5～10 minutes time; Adding serum-free DMEM 3～10ml centrifugal centrifugal condition is 1000～3000 rev/mins, 5～10 minutes time.
The main raw and the reagent that relate among the present invention are selected from:
GOLDKEY nucleic acid and protein analysis software and nucleic acid and Protein Data Bank CD are provided by the fundamental research of Chinese Military Medical Science Institute.Serous cystadenocarcinoma of ovary clone OVCAR-3, ovarian serous papillary cystic adenocarcinoma cell SKOV-3, ovarian epithelial cancer cells 3AO is that this laboratory is frozen, the SP2/0 myeloma cell strain is that international peace hospital of PLA test center is frozen, (in mouse 6～8 weeks of age, heavily about 18～25g) test animal center available from the Hebei Medical University academic test (examination) to BALB/c mouse.DMEM substratum, RPMI 1640 substratum are U.S. GIBCO company product, and foetal calf serum is a Hangzhou folium ilicis chinensis company product.Mouse SP detects and DAB colouring reagents box is purchased company of China fir Golden Bridge in Beijing.Protein extraction test kit and proteinmarker are match Parkson company product, and the Xylene Brilliant Cyanine G protein detection kit is that bio-engineering research institute product is built up in Nanjing, and the monoclonal antibody parting kit is purchased the Boehriger company in the U.S..
The evaluation of anti-human soluble mesothelin related protein monoclonal antibody
(1) titration
Utilize the compound peptide antigen of soluble mesothelin (1mg/ml) 1: 50～4000 dilution bags by microwell plate, utilize euzymelinked immunosorbent assay (ELISA) to detect antibody titer.
(2) the antibody subclass is measured
With monoclonal antibody parting kit (Boehriger company), press the operation of test kit specification sheets.
(3) specificity is identified
1. SP immunocytochemical method
Cover glass is positioned in aseptic 6 well culture plates, and the Proliferation of Human Ovarian Cell that adds logarithmic phase is OVCAR-3, SKOV3,3AO, makes cell climbing sheet.Adopt streptavidin peroxidase (SP) connection method, detect the monoclonal antibody of preparation and the specificity of native protein reaction.It is positive brown yellow granule to occur with after birth.More than test triplicate.
2. the immune marking detects the specificity of monoclonal antibody
Collect OVCAR-3 respectively, 3AO, 3 groups of cells such as SKOV3, collect cell protein with the protein extraction test kit, the Xylene Brilliant Cyanine G protein detection kit carries out quantitatively each histone of collecting, get equal protein (50 μ g) row sodium laurylsulfonate-polyphenyl alkene acrylamide gel electrophoresis, albumen is transferred on the pvdf membrane, add one and anti-ly [be respectively anti-human soluble mesothelin related protein monoclonal antibody (clone K1, U.S. ZYMED company product, extent of dilution is 1: 150) and adopt the anti-human soluble mesothelin related protein monoclonal antibody of present method preparation, code name 2H10 (extent of dilution is 1: 200)].Blank replaces one to resist with PBS.More than test triplicate.
The result
1, the multi-parameter prediction result of the plain B cell antigen epi-position of people's mesothelium
As shown in Figure 1, be higher than the epitope that the pairing peptide section of the amino-acid residue of predicting thresholding is prediction; Fig. 2 shows antigenic secondary structure prediction result; Fig. 3 is a Wu Shi antigenicity index analysis curve.According to the result of above-mentioned analysis, prediction has the peptide section of epitope to be summarized in table 1.As can be seen from the table, the position of using the peptide section that a plurality of epitope numbers that different Forecasting Methodology predicts and epitope may occur is different, but in aminoacid sequence 153-162,282-292 and three peptide sections of 471-481, multiple Forecasting Methodology institute prediction result unanimity, wetting ability and accessibility are preferably arranged, on secondary structure, contain the irregular of easy formation epitope and curl and β-corner structure.Therefore, the B cell antigen epi-position of soluble mesothelin may be positioned at this zone or they near.Wherein the 297-600 aminoacid sequence is a soluble mesothelin, and we select to have synthesized the plain molecule N-terminal of mesothelium amino-acid residue 471-481 fragment, contain 11 amino-acid residues, and order is Q D L D T C D P R Q L.
Forecasting Methodology prediction epi-position
Antigenicity parameter 142-151,153-165,268-280,306-315,471-480,560-571
Wetting ability parameter 153-165,218-229,271-280,290-300,408-419,425-438,
471-480，557-567
Accessibility parameter 153-165,218-229,271-280,280-290,402-417,473-485,
560-570
Snappiness parameter 153-162,219-225,279-291,297-310,405-416,471-483,
557-568
Secondary structure (β-corner) 133,155,193,213,236,282,302,412,477,594
Wu Shi prediction procedure 153-167,218-229,271-283,285-295,303-315,409-419,
470-482，571-580
2, it is synthetic that synthetic 11 amino-acid residues to N-terminal 471-481 in the plain sequence of people's mesothelium of the plain B cell epitope of people's mesothelium antigen peptide have carried out small peptide, antigen peptide combined with KLH form compound peptide antigen, is faint yellow solid, with 1～3mg immune mouse.
3, anti-people SMR MONOCLONAL ANTIBODIES SPECIFIC FOR
Use SMR-KLH as immunogen, adopt repeatedly immune mouse of subcutaneous entrapping method, obtained to secrete the mouse boosting cell of anti-SMR antibody, mouse boosting cell can merge external with SP2/0 myeloma cell preferably under the inducing of PEG, form hybridoma cell strain, through the indirect enzyme-linked immunosorbent method, filter out hybridoma 2 strains of anti-SMR polypeptide antibody, be respectively 2H10,6D9, through going down to posterity and freeze thawing the 2H10 and the 6D9 strain of energy stably excreting antibody repeatedly, enlarged culturing, the preparation mouse ascites produces anti-SMR monoclonal antibody, and antibody titer is 1: 102400.The IgG subclass of monoclonal antibody (2H10,6D9) is IgG1 (heavy chain), and light chain is the κ chain.
4, the specificity of antibody is identified
(1) immunocytochemistry streptomycete avidin-superoxide method detected result
Confirm that through the plain antibody of commercial anti mesothelium (5B2) it is plain positive that ovarian cancer cell line OVCAR-3, SKOV3,3AO are mesothelium, sees Fig. 4.The anti-SMR antibody 2H10 of this test preparation and 6D9 can with above-mentioned 3 kinds of cell responses, all visible significantly brown yellow granule calmness on cytolemma is seen Fig. 7.
(2) immune marking detected result
Polypeptide is all obtained single band at antibody 2H10 and 6D9 through S sodium laurylsulfonate-polyphenyl alkene acrylamide gel analysis with discussing to sell the plain antibody K1 of anti-mesothelium and dye respectively about molecular weight 40kD, see Fig. 8.
Substantive distinguishing features that the present invention is obtained and significant technical progress are:
(mesothelin MSLN) carries out the prediction of B-cell epitope to appliance computer to the mesothelium element by integrated forecasting.Synthetic corresponding peptides section and immune BALB/c mouse adopt hybridoma technology to prepare monoclonal antibody, utilize immunocytochemistry and immune marking analysis that prepared antibody is identified.The epitope of integrated forecasting methods analyst mesothelium element may be near 153-162,282-292 and 471-481 amino-acid residue or its, selectivity has been synthesized the highest soluble mesothelin related protein epi-position that is positioned at the 471-481 amino-acid residue of possibility, prepared anti-human soluble mesothelin related protein monoclonal antibody (2H10 and 6D9), analyzed this antibody through immunocytochemistry and the immune marking and have higher specificity.Lay a good foundation for further utilizing enzyme linked immunological that soluble mesothelin related protein is detected.
Description of drawings
Accompanying drawing of the present invention has:
Fig. 1 is antigenicity parameter predicting the outcome to the plain sequence of mesothelium.
Fig. 2 is wetting ability parameter predicting the outcome to the plain sequence of mesothelium.
Fig. 3 is accessibility parameter predicting the outcome to the plain sequence of mesothelium.
Fig. 4 snappiness parameter predicts the outcome to the plain sequence of mesothelium.
Fig. 5 is the secondary structure prediction of the plain aminoacid sequence of mesothelium.
Fig. 6 is Wu Shi antigenic index analytic curve predicting the outcome to MSLN sequence epitope.
Fig. 7 is the immunocytochemistry result of mouse-anti human soluble mesothelin monoclonal antibody specificity analyses.
A in this accompanying drawing: react with antibody 5B2; B: react with antibody 2H10
1: ovarian cancer cell OUCAR3; 2: ovarian cancer cell SKOV3; 3: ovarian cancer cell 3AO
Fig. 8 is the immune marking detected result analysis of mouse-anti human soluble mesothelin antibodies specific
A in this accompanying drawing: react with antibody K1; B: react with antibody 2H10
M: protein labeling; 1:OVCAR-3 albumen swimming lane; 2:SKOV3 albumen swimming lane;
3:3AO albumen swimming lane.
Embodiment
Below in conjunction with embodiment the present invention is further described, but not as limitation of the invention further.
Anti-human soluble mesothelin related protein MONOCLONAL ANTIBODIES SPECIFIC FOR method comprises following processing step:
The multi-parameter prediction and the antigen peptide of A, the plain B cell antigen epi-position of people's mesothelium are synthetic
(1) one-parameter is predicted respectively
Use the plain aminoacid sequence total length of GOLDKEY software analysis mesothelium and analyze the plain aminoacid sequence total length of mesothelium, use Hopp ﹠amp; Woods wetting ability parameter, Welling antigenicity parameter, accessibility parameter, secondary structure are carried out the one-parameter prediction respectively to epitope, and the peak that all is higher than thresholding is the site that dopes; Wherein the plain aminoacid sequence total length of mesothelium is as follows:
1 malptarpll?gscgtpalgs?llfllfslgw?vqpsrtlage?tgqeaapldg?vlanppniss
61 lsprqllgfp?caevsglste?rvrelavala?qknvklsteq?lrclahrlse?ppedldalpl
121?dlllflnpda?fsgpqactrf?fsritkanvd?llprgaperq?rllpaalacw?gvrgsllsea
181?dvralgglac?dlpgrfvaes?aevllprlvs?cpgpldqdqq?eaaraalqgg?gppygppstw
241?svstmdalrg?llpvlgqpii?rsipqgivaa?wrqrssrdps?wrqpertilr?prfrrevekt
301?acpsgkkare?ideslifykk?weleacvdaa?llatqmdrvn?aipftyeqld?vlkhkldely
361?pqgypesviq?hlgylflkms?pedirkwnvt?sletlkalle?vnkghemspq?aprrplpqva
421?tlidrfvkgr?gqldkdtldt?ltafypgylc?slspeelssv?ppssiwavrp?qdldtcdprq
481?ldvlypkarl?afqnmngsey?fvkiqsflgg?aptedlkals?qqnvsmdlat?fmklrtdayl
541?pltvaevqkl?lgphveglka?eerhrpvrdw?ilrqrqddld?tlglglqggi?pngylvldls
601?mqealsgtpc?llgpgpvltv?lalllas
Above-mentioned aminoacid sequence is taken passages from NCBI protein sequence storehouse
(2) integrated forecasting
Use the method contrast in antigenicity index analysis curve and the above-mentioned steps (1), to avoid error, the aminoacid sequence of finally having determined antigen site is Q D L D T C D P R Q L;
(3) antigen peptide is synthetic
Utilize tyrosine to have the special construction of two amino, synthesize earlier and have 4 aminoterminal tyrosine cores, peptide chain extends to the N end from the C end, has been to press previously selected aminoacid sequence synthetic (synthetic by the polypeptide lab A BI433A of Xuan Wu, Beijing hospital type polypeptide automatic DNA synthesizer DNA) with each amino;
B, mouse-anti people SMR MONOCLONAL ANTIBODIES SPECIFIC FOR
(1) mouse boosting cell preparation
Nitrocellulose filter is made diaphragm, soaked overnight in containing the antigenic solution of the compound peptide of SMR, the subcutaneous immune BALB/c mouse in first back, after behind the initial immunity 10～14 days, 20～28 days, carry out booster immunization twice, after the last immunity 3～10 days, under aseptic condition, take out spleen, grinding makes splenocyte free, adopt GKN damping fluid washing back centrifugal, sedimentary cell is resuspended with 10～30ml serum-free DMEM nutrient solution, and it is stand-by to put into CO2gas incubator;
(2) myeloma cell's preparation
SP2/0 myeloma cell strain elder generation before fusion does to adapt to the substratum that contains the 8-azaguanine cultivates, and getting the mouse peritoneal cell is feeder cell, and the adjustment cell concn is 1～3 * 10
5/ ml placed culture plate to cultivate in 24～48 hours in advance; Cytogamy same day, collect the SP2/0 cell that is in logarithmic phase, add serum-free DMEM substratum 10～30ml carry out centrifugal, with 10～20ml serum-free DMEM substratum resuspension cell, the mensuration cytoactive;
(3) cytogamy
With myeloma cell and splenocyte by 1: 7～10 mixed, with serum-free medium washing, centrifugal back supernatant discarded, at room temperature splenocyte and SP2/0 myeloma cell are merged, polyoxyethylene glycol 0.5～3ml the effect 0.5～3 minute that adds 37 ℃ again, add serum-free medium 0.5～10ml, end the polyoxyethylene glycol effect, it is centrifugal to add serum-free DMEM 3～10ml, removes supernatant; Add the HAT substratum that contains 20% bovine serum, make cell reach 0.5～10 * 10
6/ ml; Add and to contain in the culture plate of feeder cell, in 10～200 μ l/ holes, 37 ℃, 5% carbonic acid gas incubator, cultivate;
(4) detect antibody titer
Detect the positive colony that each hole produces anti-SMR antibody with the indirect enzyme-linked immunosorbent method;
(5) a large amount of preparations of antibody
Adopting the gauge dilution method to carry out mono-clonal to the positive colony cell cultivates; The mouse peritoneal injection is adopted in a large amount of preparations of antibody, prior to BALB/c mouse abdominal injection whiteruss, and 2～4 all pneumoretroperitoneum injections 1～10 * 10
6Individual hybridoma, inoculating cell was collected ascites after 10～15 days, and the centrifuging and taking supernatant liquor is frozen in refrigerator.
Technical qualification in the present embodiment in each processing step are:
In the step in the process B (1), it is 0.2～0.5cm * 0.5～1.0cm that nitrocellulose filter is made the diaphragm-operated specification.
In the step in the process B (1), the subcutaneous immune BALB/c mouse in first back adopts entrapping method.
In the step in the process B (1), centrifugal condition is 1000～3000 rev/mins, 5～10 minutes time, abandons supernatant, and repeated centrifugation once.
In the step in the process B (1), the method for booster immunization is identical with the method for initial immunity.
In the step in the process B (2), culture plate is 96 holes, and centrifugal condition is 1000～3000 rev/mins, 5～10 minutes time, 3 times; The method of measuring cytoactive is trypan blue dyeing.
In the step in the process B (3), be 500～3000 rev/mins with serum-free medium washing, centrifugal condition in centrifugal, 5～10 minutes time; Adding serum-free DMEM 3～10ml centrifugal centrifugal condition is 1000～3000 rev/mins, 5～10 minutes time.
The main raw and the reagent that relate in the present embodiment are selected from:
GOLDKEY nucleic acid and protein analysis software and nucleic acid and Protein Data Bank CD are provided by the fundamental research of Chinese Military Medical Science Institute.Serous cystadenocarcinoma of ovary clone OVCAR-3, ovarian serous papillary cystic adenocarcinoma cell SKOV-3, ovarian epithelial cancer cells 3AO is that this laboratory is frozen, the SP2/0 myeloma cell strain is that international peace hospital of PLA test center is frozen, (in mouse 6～8 weeks of age, heavily about 18～25g) test animal center available from the Hebei Medical University academic test (examination) to BALB/c mouse.DMEM substratum, RPMI 1640 substratum are U.S. GIBCO company product, and foetal calf serum is a Hangzhou folium ilicis chinensis company product.Mouse SP detects and DAB colouring reagents box is purchased company of China fir Golden Bridge in Beijing.Protein extraction test kit and proteinmarker are match Parkson company product, and the Xylene Brilliant Cyanine G protein detection kit is that bio-engineering research institute product is built up in Nanjing, and the monoclonal antibody parting kit is purchased the Boehriger company in the U.S..
Claims (7)
1, anti-human soluble mesothelin related protein MONOCLONAL ANTIBODIES SPECIFIC FOR method comprises following processing step:
The multi-parameter prediction and the antigen peptide of A, the plain B cell antigen epi-position of people's mesothelium are synthetic
(1) one-parameter is predicted respectively
Analyze the plain aminoacid sequence total length of mesothelium, use Hopp ﹠amp; Woods wetting ability parameter, Welling antigenicity parameter, accessibility parameter, secondary structure are carried out the one-parameter prediction respectively to epitope, and the peak that all is higher than thresholding is the site that dopes; Wherein the plain aminoacid sequence total length of mesothelium is as follows:
1 malptarpll?gscgtpalgs?llfllfslgw?vqpsrtlage?tgqeaapldg?vlanppniss
61 lsprqllgfp?caevsglste?rvrelavala?qknvklsteq?lrclahrlse?ppedldalpl
121?dlllflnpda?fsgpqactrf?fsritkanvd?llprgaperq?rllpaalacw?gvrgsllsea
181?dvralgglac?dlpgrfvaes?aevllprlvs?cpgpldqdqq?eaaraalqgg?gppygppstw
241?svstmdalrg?llpvlgqpii?rsipqgivaa?wrqrssrdps?wrqpertilr?prfrrevekt
301?acpsgkkare?ideslifykk?weleacvdaa?llatqmdrvn?aipftyeqld?vlkhkldely
361?Pqgypesviq?hlgylflkms?pedirkwnvt?sletlkalle?vnkghemspq?aprrplpqva
421?tlidrfvkgr?gqldkdtldt?ltafypgylc?slspeelssv?ppssiwavrp?qdldtcdprq
481?ldvlypkarl?afqnmngsey?fvkiqsflgg?aptedlkals?qqnvsmdlat?fmklrtdavl
541?pltvaevqkl?lgphveglka?eerhrpvrdw?ilrqrqddld?tlglglqggi?pngylvldls
601?mqealsgtpc?llgpgpvltv?lalllas
(2) integrated forecasting
Use the method contrast in antigenicity index analysis curve and the above-mentioned steps (1), to avoid error, the aminoacid sequence of finally having determined antigen site is Q D L D T C D P R Q L;
(3) antigen peptide is synthetic
Utilize tyrosine to have the special construction of two amino, synthetic earlier have 4 aminoterminal tyrosine cores, and peptide chain extends to the N end from the C end, has been to press previously selected aminoacid sequence to synthesize with each amino;
B, anti-people SMR MONOCLONAL ANTIBODIES SPECIFIC FOR
(1) mouse boosting cell preparation
Nitrocellulose filter is made diaphragm, soaked overnight in containing the antigenic solution of the compound peptide of SMR, the subcutaneous immune BALB/c mouse in first back, after behind the initial immunity 10～14 days, 20～28 days, carry out booster immunization twice, after the last immunity 3～10 days, under aseptic condition, take out spleen, grinding makes splenocyte free, adopt GKN damping fluid washing back centrifugal, sedimentary cell is resuspended with 10～30ml serum-free DMEM nutrient solution, and it is stand-by to put into CO2gas incubator;
(2) myeloma cell's preparation
SP2/0 myeloma cell strain elder generation before fusion does to adapt to the substratum that contains the 8-azaguanine cultivates, and getting the mouse peritoneal cell is feeder cell, and the adjustment cell concn is 1～3 * 10
5/ ml placed culture plate to cultivate in 24～48 hours in advance; Cytogamy same day, collect the SP2/0 cell that is in logarithmic phase, add serum-free DMEM substratum 10～30ml carry out centrifugal, with 10～20ml serum-free DMEM substratum resuspension cell, the mensuration cytoactive;
(3) cytogamy
With myeloma cell and splenocyte by 1: 7～10 mixed, with serum-free medium washing, centrifugal back supernatant discarded, at room temperature splenocyte and SP2/0 myeloma cell are merged, PEG 0.5～3ml the effect 0.5～3 minute that adds 37 ℃ again, add serum-free medium 0.5～10ml, end the PEG effect, it is centrifugal to add serum-free DMEM 3～10ml, removes supernatant; Add the HAT substratum that contains 20% bovine serum, make cell reach 0.5～10 * 10
6/ ml; Add and to contain in the culture plate of feeder cell, in 10～200 μ l/ holes, 37 ℃, 5% carbonic acid gas incubator, cultivate;
(4) detect antibody titer
Detect the positive colony that each hole produces anti-SMR antibody with indirect elisa method;
(5) a large amount of preparations of antibody
Adopting the gauge dilution method to carry out mono-clonal to the positive colony cell cultivates; The mouse peritoneal injection is adopted in a large amount of preparations of antibody, prior to BALB/c mouse abdominal injection whiteruss, and 2～4 all pneumoretroperitoneum injections 1～10 * 10
6Individual hybridoma, inoculating cell was collected ascites after 10～15 days, and the centrifuging and taking supernatant liquor is frozen.
2, anti-human soluble mesothelin related protein MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 1 is characterized in that in the step (1) in the described process B, it is 0.2～0.5cm * 0.5～1.0cm that nitrocellulose filter is made the diaphragm-operated specification.
3, anti-human soluble mesothelin related protein MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 1 is characterized in that in the step (1) in the described process B, and the subcutaneous immune BALB/c mouse in first back adopts entrapping method.
4, anti-human soluble mesothelin related protein MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 1, it is characterized in that in the step (1) in the described process B, centrifugal condition is 1000～3000 rev/mins, 5～10 minutes time, abandons supernatant, and repeated centrifugation once.
5, according to claim 1 or 3 described anti-human soluble mesothelin related protein MONOCLONAL ANTIBODIES SPECIFIC FOR methods, it is characterized in that the method for booster immunization is identical with the method for initial immunity in the step (1) in the described process B.
6, anti-human soluble mesothelin related protein MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 1, it is characterized in that in the step (2) in the described process B, culture plate is 96 holes, and centrifugal condition is 1000～3000 rev/mins, 5～10 minutes time, 3 times; The method of measuring cytoactive is trypan blue dyeing.
7, anti-human soluble mesothelin related protein MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 1, it is characterized in that in the step (3) in the described process B, with serum-free medium washing, centrifugal condition in centrifugal is 500～3000 rev/mins, 5～10 minutes time; Adding serum-free DMEM 7ml centrifugal centrifugal condition is 1000～3000 rev/mins, 5～10 minutes time.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710061497 CN101012280A (en) | 2007-02-07 | 2007-02-07 | Method of preparing monoclonal antibody against human soluble mesothelin related protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710061497 CN101012280A (en) | 2007-02-07 | 2007-02-07 | Method of preparing monoclonal antibody against human soluble mesothelin related protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101012280A true CN101012280A (en) | 2007-08-08 |
Family
ID=38700020
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200710061497 Pending CN101012280A (en) | 2007-02-07 | 2007-02-07 | Method of preparing monoclonal antibody against human soluble mesothelin related protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101012280A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102243239A (en) * | 2011-04-25 | 2011-11-16 | 重庆大学 | Quick colloidal gold detection test strip for xanthomonas citri |
| CN102618940A (en) * | 2011-01-31 | 2012-08-01 | 艾比玛特生物医药（上海）有限公司 | Antibody preparation method and obtained antibody and antibody database |
| CN101951946B (en) * | 2007-10-01 | 2014-12-10 | 百时美施贵宝公司 | Human antibodies that bind mesothelin, and uses thereof |
| CN104558170A (en) * | 2014-12-22 | 2015-04-29 | 广西大学 | Preparation process of antivenin |
| CN107850605A (en) * | 2015-05-14 | 2018-03-27 | 爱科谱迅病理研究公司 | The SRM/MRM measure of mesothelin (MSLN) protein |
| CN109705219A (en) * | 2019-01-08 | 2019-05-03 | 上海霖科生物科技有限公司 | Monoclonal antibody in conjunction with mesothelin and preparation method thereof |
| CN110698562A (en) * | 2019-10-31 | 2020-01-17 | 浙江蓝盾药业有限公司 | Anti-human MSLN monoclonal antibody |
| CN111247249A (en) * | 2017-10-31 | 2020-06-05 | 国立大学法人冈山大学 | DNA, polypeptide, anti-mesothelin antibody, tumor imaging agent and complex |
| CN113490510A (en) * | 2019-01-08 | 2021-10-08 | 美国政府(由卫生和人类服务部的部长所代表) | Cross-species single domain antibodies targeting mesothelin for the treatment of solid tumors |
-
2007
- 2007-02-07 CN CN 200710061497 patent/CN101012280A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101951946B (en) * | 2007-10-01 | 2014-12-10 | 百时美施贵宝公司 | Human antibodies that bind mesothelin, and uses thereof |
| CN102618940A (en) * | 2011-01-31 | 2012-08-01 | 艾比玛特生物医药（上海）有限公司 | Antibody preparation method and obtained antibody and antibody database |
| WO2012103797A1 (en) * | 2011-01-31 | 2012-08-09 | 艾比玛特生物医药（上海）有限公司 | Antibody preparation method, and antibody and antibody library thus prepared |
| US9511342B2 (en) | 2011-01-31 | 2016-12-06 | Abmart (Shanghai) Co., Ltd. | Antibody preparation method, and antibody and antibody library thus prepared |
| CN102243239A (en) * | 2011-04-25 | 2011-11-16 | 重庆大学 | Quick colloidal gold detection test strip for xanthomonas citri |
| CN104558170A (en) * | 2014-12-22 | 2015-04-29 | 广西大学 | Preparation process of antivenin |
| CN107850605A (en) * | 2015-05-14 | 2018-03-27 | 爱科谱迅病理研究公司 | The SRM/MRM measure of mesothelin (MSLN) protein |
| CN111247249A (en) * | 2017-10-31 | 2020-06-05 | 国立大学法人冈山大学 | DNA, polypeptide, anti-mesothelin antibody, tumor imaging agent and complex |
| US11975080B2 (en) | 2017-10-31 | 2024-05-07 | National University Corporation Okayama University | Anti-mesothelin polypeptide, and tumor imaging agents and complexes thereof |
| CN109705219A (en) * | 2019-01-08 | 2019-05-03 | 上海霖科生物科技有限公司 | Monoclonal antibody in conjunction with mesothelin and preparation method thereof |
| CN109705219B (en) * | 2019-01-08 | 2020-11-13 | 上海霖科生物科技有限公司 | Monoclonal antibody combined with mesothelin and preparation method thereof |
| CN112321714A (en) * | 2019-01-08 | 2021-02-05 | 上海霖科生物科技有限公司 | Monoclonal antibody combined with mesothelin and variable region coding sequence thereof |
| CN113490510A (en) * | 2019-01-08 | 2021-10-08 | 美国政府(由卫生和人类服务部的部长所代表) | Cross-species single domain antibodies targeting mesothelin for the treatment of solid tumors |
| CN110698562A (en) * | 2019-10-31 | 2020-01-17 | 浙江蓝盾药业有限公司 | Anti-human MSLN monoclonal antibody |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101012280A (en) | Method of preparing monoclonal antibody against human soluble mesothelin related protein | |
| KR101485202B1 (en) | T cell epitope databases | |
| CN103819561A (en) | Anti-CD24 (Cluster of Differentiation 24) monoclonal antibody, and variable region sequence and application thereof | |
| CN116731181B (en) | Anti-human CD10 protein rabbit monoclonal antibody and application thereof | |
| CN107850596A (en) | Method for detecting tissue infiltration's NK cells | |
| CN113621069B (en) | anti-HER-2 protein monoclonal antibody, and preparation method and application thereof | |
| CN104448001A (en) | Anti-5-methylcytosine monoclonal antibody and application thereof | |
| Axelrod et al. | Idiotypic cross-reactivity of immunoglobulins expressed in Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia, and mantle zone lymphocytes of secondary B-cell follicles | |
| CN105017403B (en) | The preparation and its application of cancer of pancreas related polypeptide DAP44 monoclonal antibodies | |
| CN105646713B (en) | A kind of monoclonal antibody and its application | |
| CN105646712B (en) | Monoclonal antibody and its application | |
| CN108484770B (en) | Recombinant rat anti-mouse CD4 monoclonal antibody, preparation method and application | |
| US11426462B2 (en) | Monovalent anti-CD3 adjuvants | |
| CN117467004B (en) | Anti-human calomelin rabbit monoclonal antibody and application thereof | |
| CN117384291B (en) | Anti-human SALL4 rabbit monoclonal antibody, preparation method and application thereof, polynucleotide molecule, expression vector and host cell | |
| CN113717279B (en) | m6Recombinant rabbit monoclonal antibody A and preparation method thereof | |
| CN117025547B (en) | Hybridoma cell strain for producing anti-B7H 3 monoclonal antibody and application thereof | |
| CN117487008B (en) | Anti-human Lin28A protein rabbit monoclonal antibody and application thereof | |
| CN116836289B (en) | Rabbit monoclonal antibody for human MPO protein and application thereof | |
| CN110054676B (en) | Immunogenic polypeptide, anti-TTC 36 antibody AP3-5 and application | |
| CN111705039B (en) | Hybridoma cell strain secreting anti-SOX 17 monoclonal antibody and application | |
| CN117384291A (en) | Anti-human SALL4 rabbit monoclonal antibody, preparation method and application thereof, polynucleotide molecule, expression vector and host cell | |
| Hayat | Antigens and antibodies | |
| CN108727496A (en) | The anti-human p63 protein monoclonal antibodies of mouse prepare and its immunohistochemistry purposes | |
| CN117821399A (en) | Mouse anti-human CDCA7 monoclonal antibody and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Open date: 20070808 |