CN1396182A - Human monoclonal antibody of human endophloeodal growth factor receptor 2 protein and its preparing process - Google Patents
Human monoclonal antibody of human endophloeodal growth factor receptor 2 protein and its preparing process Download PDFInfo
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Abstract
A humanized monoclonal antibody of human endophloeodal growth factor receptor 2 protein is prepared from enternal segment of HER2/neucell through such steps as culturing the B cells of the person sensitive to HER2; fusing the said B cells with human osteoma cells (karpas 707 H); screening the external segment monoclonal anti body against HER2 cell; and amplification of the said monoclonal antibody. It features that antigen epitope is the external segment polypeptide of HER2/neu cell for specifically binding the external segment of HER2/neu cell, and the all gene sequences for syntehsis come from human B lymph cells and human osteoma cells.
Description
Technical field:
The present invention relates to biomedical sector, relate to a kind of people's endothelial growth factor receptor 2 albumen total man monoclonal antibodies and preparation method thereof or rather.
Background technology:
People's endothelial growth factor receptor 2 albumen (HER-2/neu albumen) are that molecular weight is 185kD, thereby claims P185 again by the strand transmembrane glycoprotein of the proto-oncogene HER-2/neu coding that is positioned at No. 17 karyomit(e) q11-22 position of people.The cell of many malignant tumours such as mammary cancer, ovarian cancer, lung cancer, cancer of the stomach, colorectal carcinoma often can detect HER-2/neu gene amplification or protein overexpression, so the HER-2/neu protein overexpression is relevant with the generation of tumour.When HER-2/neu albumen during in the cell surface overexpression, the heterodimer of HER-2/neu acceptor forms quantity and increases, and the cell signal transfer system can overactivity, thereby causes uncontrolled cellular proliferation, and canceration takes place.Because HER-2/neu albumen is positioned at cell surface, easily approaching by antibody, thereby, be people mouse mosaic antibody at the Herceptin that uses in the market at the design of HER-2/neu albumen for the conduction of blocking-up cell proliferative signal can prepare monoclonal antibody at HER-2/neu.Yet there is mouse IgG complementary determining region (CDR) in the mouse monoclonal antibody, thereby have a significant side effects---the anti-rat immune globulin reaction of people (HAMA), HAMA can seriously limit interior application of body of monoclonal antibody, significantly reduce the result of treatment of mouse resource monoclonal antibody, especially those need the mouse monoclonal antibody used in the iterative, HAMA to influence meeting stronger.In order to overcome the HAMA reaction, the humanization technology of mouse resource monoclonal antibody constantly is born with ripe, as people-mouse chimeric antibody, reshaping antibody and antibody library technology etc.Though the monoclonal antibody of utilizing above-mentioned technology to produce has reduced the HAMA reaction to a great extent,, therefore can not fundamentally solve the HAMA problem owing to still have mouse IgG CDR in the monoclonal antibody molecule.
Summary of the invention:
In order fundamentally to solve the HAMA problem, improve result of treatment, the invention provides a kind of people's endothelial growth factor receptor 2 albumen total man monoclonal antibodies, a kind of people's endothelial growth factor receptor 2 albumen total man MONOCLONAL ANTIBODIES SPECIFIC FOR methods are provided simultaneously.
Technical solution provided by the invention is as follows: a kind of people's endothelial growth factor receptor 2 albumen total man monoclonal antibodies, and its special character is that it is made by following preparation method, may further comprise the steps successively,
1. cultivate HER2 sensitization human B cell;
2. the human B cell of HER2 sensitization and people's osteoma cytogamy, specific operation process is as follows: at first will
(1～2) * 10 that are used to merge
6Human myeloma cell Karpas 707H and step are 1. resultant
(3～7) * 10
6The sensitization human B cell mixes, and adds dropwise in 2 minutes that 1～2ml is fresh to be joined
The PEG-2000 of system; Splash into RPMI1640 50～60ml then fast, centrifugal 5 minutes of 200 * g
Clock is abandoned supernatant, repeats to add RPMI1640 50～60ml washing; At last fused cell is transferred
Make 1～2 * 10
6/ ml cultivated feeder cell 1 day in advance, and every hole adds in Tissue Culture Plate
100ul, HAT RPMI1640 cultivated 12～15 days, changeed HT RPMI1640 and cultivated 7～12
My god;
The screening of 3. anti-HER2 extracellular fragment monoclonal antibody;
The amplification of 4. anti-HER2 extracellular fragment monoclonal antibody.
A kind of people's endothelial growth factor receptor 2 albumen total man MONOCLONAL ANTIBODIES SPECIFIC FOR methods may further comprise the steps successively:
1. cultivate HER2 sensitization human B cell;
2. the human B cell of HER2 sensitization and people's osteoma cytogamy, specific operation process is as follows: at first will
Be used to merge 1～2 * 10
6Karpas 707H and step 1. resulting 3～7 * 10
6Sensitization people
The B cytomixis dropwise added the freshly prepared PEG-2000 of 1～2ml in 2 minutes; Quick then
Splash into RPMI1640 50～60ml, centrifugal 5 minutes of 200 * g abandons supernatant, repeats to add
RPMI1640 50～60ml washing; At last fused cell is modulated into 1～2 * 10
6/ ml, the pre-cultivation carefully
Born of the same parents, every hole adds convention amount in Tissue Culture Plate, and HAT RPMI1640 cultivated 12～15 days,
Changeing HT RPMI1640 cultivated 7～12 days;
The screening of 3. anti-HER2 extracellular fragment monoclonal antibody;
The amplification of 4. anti-HER2 extracellular fragment monoclonal antibody.
In the such scheme, described step 1., can realize by three approach: article one approach is directly to extract the human B cell of HER2 sensitization from patient with breast cancer's blood of high expression level HER2/neu, at first determines the target volunteer, gets patient's peripheral blood 2～5ml, measure Anti-HER 2 with commercial kit, select its titre greater than 1: 64, take out peripheral blood 50～100ml from the target volunteer then, add B cellular segregation liquid, 200 * g is centrifugal, gets middle level oyster white B cellular layer; The second approach is at first to get high expression level HER2/neu patient with breast cancer's cancer week lymphoglandula, aseptic technique shreds, tryptic digestion 8～12 hours, cross 150～200 order stainless steel sifts, be suspended on the 20ml lymphocyte layering liquid, 200 * g is centrifugal, collects interface oyster white lymphocyte, and the bone-marrow-derived lymphocyte that will obtain in order to last method is tested with commercialization immunobead (anti-human IgG antibody of mark and anti-human IgM antibody) and whether confirmed sensitization then; Article three, approach is external HER2 immunity, with the human peripheral blood B cell of last sensitization after conditioned medium is cultivated 24 hours, change in the perfect medium and cultivated 5～7 days, the composition of described perfect medium contains HER2/neu 1～2ng/ml, AB type human serum 1～10%, interleukin (IL)-2 100～150U/ml, IL-4 8～12ng/ml, IL-6 16～20ng/ml, scavenger cell 1～2 * 10
5/ ml.
3. step in the such scheme at first will be used for the affine pure HER2/neu bag quilt of the microwell plate of screening and cloning with purchase, and every hole adds the convention amount cells and supernatant, and 37 ℃ of cultivations are spent the night; The mouse-anti human IgG Fc mAb that adds the HRP mark then after the washing cultivated 30～45 minutes for 37 ℃, added substrate TMB colour developing after the washing, and the OD value is positive greater than 10 * blank OD person.
4. the step of such scheme is that the positive colony hybridoma is adjusted to 1～2 * 10
6/ ml gets 1ml and is expelled to nude mice abdominal cavity or SCID mouse peritoneal, gets ascites after 7～10 days, and 200～250 * g is centrifugal, gets supernatant, adds 30～50% glycerol, preserves below-20 ℃; Or the positive colony hybridoma is adjusted to 1～2 * 10
6/ ml gets the 20ml aseptic technique and adds in the bio-reactor, cultivates about 2 weeks, and according to the antibody titers of cell treatment system situation and sampling and measuring, it is centrifugal to get nutrient solution 200～250 * g, gets supernatant, adds 30～50% glycerol, preserves below-20 ℃.
5. such scheme also comprises step: purification step, what this purification step adopted is affinity chromatography.
5. above-mentioned steps is people HER2/neu antigen extracellular fragment to be coupled to Sephrose 4B through CNBr prepare total man HER2/neu monoclonal antibody affinity chromatography post, described total man HER2/neu monoclonal antibody affinity chromatography post 0.01M PB, pH6.8,0.1mM the Buffer A pre-equilibration of α-mercaptothanol, 4 ℃ of preservations, its operation is the supernatant that at first 100ml is contained total man HER2/neu monoclonal antibody, add in the affinity column of 200ml column volume, 4 ℃ are spent the night, then with Buffer A fully stream wash OD280 less than 0.01, flow velocity is 8ml/min, uses 0.01M PB, pH6.8,0.1mM α-mercaptothanol, 150mM NaClBuffer B wash-out is collected the part of OD280＞0.02 at last, lyophilize ,-20 ℃ of preservations; Or be coupled to Sephrose 4B with albumin A (SPA) through CNBr and prepare the SPA affinity column.SPA affinity column 0.2M glycine, pH2.3,0.1mM α-mercaptothanol Buffer C pre-equilibration, and stream is washed 200ml, flow velocity 0.5ml/min, its operation is the supernatant that at first 100ml is contained total man HER2/neu monoclonal antibody, adds in the affinity column of 100ml column volume, and 4 ℃ are spent the night, use 0.01M PB then, pH8.0,0.1mM α-mercaptothanol Buffer D fully stream washes OD280 less than 0.02, flow velocity 1ml/min, use the 0.1M citrate buffer solution, pH6.0,0.1mM α-mercaptothanol Buffer E wash-out IgG1 uses the 0.1M citrate buffer solution, pH3.5,0.1mM α-mercaptothanol BufferF wash-out IgG2b uses the 0.1M citrate buffer solution, pH4.4,0.1mM α-mercaptothanol BufferG wash-out IgG2a, collect the part of OD280＞0.02 at last, lyophilize ,-20 ℃ of preservations; Or be coupled to Sephrose 4B with Protein G (Protein G) CNBr and prepare Protein G affinity column, ProteinG affinity column 0.2M glycine, pH2.3,0.1mM α-mercaptothanol Buffer C pre-equilibration, and stream is washed 200ml, flow velocity 0.5ml/min, its operation is the supernatant that at first 100ml is contained total man HER2/neu monoclonal antibody, adds in the affinity column of 100ml column volume, and 4 ℃ are spent the night, use 0.01MPB then, pH8.0,0.1mM α-mercaptothanol Buffer D fully stream washes OD280 less than 0.02, flow velocity 1ml/min, use the 0.1M citrate buffer solution, pH3.0,0.1mM α-mercaptothanol BufferH wash-out IgG collects the part of OD280＞0.02 at last, lyophilize ,-20 ℃ of preservations.
Compared with prior art, advantage of the present invention is:
1, do not contain any mouse source protein composition in the ER2/neu monoclonal antibody molecule, fundamentally solved the HAMA problem, but the long-range vivo medicine-feeding improves the effect of treatment;
2, be that the natural selection of human immune system produces, have and identical molecular structure of natural antibody and conformation therefore, identical immune response ability is arranged with natural antibody;
3, because complete Ig molecular structure is arranged, therefore have the longer transformation period;
4, the output than traditional monoclonal antibody expression system is higher.
Embodiment:
The invention provides a kind of people's endothelial growth factor receptor 2 albumen total man monoclonal antibodies, the characteristic of this monoclonal antibody is: epitope is a HER2/neu extracellular fragment polypeptide, can combine with the HER2/neu extracellular fragment specifically; Instruct the full gene sequence of synthesizing this monoclonal antibody all to derive from people's bone-marrow-derived lymphocyte and human myeloma cell; This monoclonal antibody and the HER2/neu extracellular fragment bonded coefficient that dissociates is equal to or less than 10
-8M; It and mouse HER2/neu extracellular fragment have cross reaction.Use this monoclonal antibody cell growth signals through HER2/neu conduction capable of blocking, thereby can be used for the clinical treatment of diseases such as numerous malignant tumours of HER-2/neu gene amplification or protein overexpression such as mammary cancer, ovarian cancer, lung cancer, cancer of the stomach, colorectal carcinoma.
Below will provide embodiment to describe people's endothelial growth factor receptor 2 albumen total man MONOCLONAL ANTIBODIES SPECIFIC FOR processes in detail, but the present invention is not limited to following embodiment.
Embodiment 1: obtain sensitization B cell preparation total man HER2/neu monoclonal antibody through peripheral blood
1. step at first determines the target volunteer, selects 100 volunteers, be the patient with breast cancer, each extracting vein blood 5ml, separation of serum,-20 ℃ of preservations, through commercialization HER2 ELISA KIT screening, the Anti-HER 2 positive and titre were the target volunteer greater than 1: 64; Get peripheral blood 50ml the target volunteer then, add lymphocyte layering liquid, 200 * g is centrifugal, gets interface B cellular layer, gets 100ul B cell and does immunobead test and judge whether sensitization of B cell.The B cell that merges sensitization is with RPMI1640 washing 3 times.2. step transfers B cell to 5 * 10 with RPMI1640
6/ ml, human myeloma cell transfers to 2 * 10 for Karpas707H RPMI
6/ ml.Karpas 707H is mixed with each 1ml of sensitization human B cell, in 2 minutes, dropwise add freshly prepared 2ml PEG-2000, splash into 50mlRPMI1640 then immediately fast and stop the PEG effect, centrifugal 5 minutes of 200 * g, abandon supernatant, repeat to add the 50mlRPMI1640 washing, fused cell is modulated into 1 * 10 with the HAT substratum
6/ ml cultivated feeder cell 1 day in advance, and every hole adds 100ul in 96 porocyte culture plates, HAT RPMI1640 cultivate 12 days (37 ℃, 5%CO
2), change HT RPMI1640 and cultivated 7 days; Step 3., the 96 hole microwell plates HER2/neu extracellular region bag quilt that is used for screening and cloning, every hole adds the 100ul cells and supernatant, 37 ℃ of cultivations are spent the night, and add the mouse-anti human IgG Fc mAb of HRP mark after the washing, cultivate 30 minutes for 37 ℃, add substrate (TMB) colour developing after the washing, the OD value is positive greater than 10 * blank OD person, and the result obtains 2 strains of anti-HER2/neu extracellular region positive colony, and standard limiting dilution assay clone obtains single clone 2E5 and 6H13; 4. step is adjusted to 1 * 10 with positive colony 2E5 hybridoma
6/ ml gets 20ml and is expelled to nude mice abdominal cavity, and every 1ml got ascites after 10 days, and surveying antibody titers is 1: 20000, and 200 * g is centrifugal, gets supernatant, and 80ml adds equivalent 50%glycerol ,-20 ℃ of preservations altogether; Step 5., personnel selection HER2/neu antigen extracellular fragment is coupled to Sephrose 4B through CNBr and prepares total man HER2/neu monoclonal antibody affinity chromatography post, total man HER2/neu monoclonal antibody affinity chromatography post 0.01M PB, pH6.8,0.1mM α-mercaptothanol Buffer A pre-equilibration 4 hours, 4 ℃ of preservations.Its operation is the supernatant that at first 160ml is contained total man HER2/neu monoclonal antibody, adds in the affinity column of 200ml column volume, and 4 ℃ are spent the night, then with Buffer A fully stream wash OD280 less than 0.01, flow velocity 8ml/min uses 0.01M PB, pH6.8,0.1mM α-mercaptothanol, 150mM NaCl Buffer B wash-out, flow velocity 2ml/min, collect the part of OD280＞0.02, lyophilize obtains the total man HER2/neu monoclonal antibody 2E5 3mg of purifying ,-20 ℃ of preservations.
Embodiment 2: obtain sensitization B cell preparation total man HER2/neu monoclonal antibody through external immunity
1. step buys the venous blood 400ml of the Trisodium Citrate anti-freezing of fresh collection from blood bank, and separated plasma is defined as the Anti-HER 2 feminine gender through commercialization HER2 ELISA KIT, adds lymphocyte layering liquid, and 200 * g is centrifugal, gets interface B cellular layer.Getting 100ul B cell does immunobead test and is defined as B cell end sensitization.The human peripheral blood B cell of end sensitization changes in the perfect medium and cultivated 5 days after conditioned medium is cultivated 24 hours, and this perfect medium contains HER2/neu extracellular region 2ng/ml, AB type human serum 1%, IL-2 100U/ml, IL-4 8ng/ml, IL-6 16ng/ml, scavenger cell 1 * 10
5/ ml defines 28%B cell sensitization through the immunobead test, and the B cell of sensitization is with RPMI1640 washing 3 times; 2. step transfers B cell to 7 * 10 with RPMI1640
6/ ml, human myeloma cell are that Karpas 707H RPMI transfers to 1 * 10
6,/ml.Karpas 707H mixes with each 1ml of sensitization human B cell, in 2 minutes, dropwise add freshly prepared 2ml PEG-2000, splash into RPMI1640 50ml then fast and stop PEG effect, centrifugal 5 minutes of 200 * g, abandon supernatant, repeat to add RPMI1640 50ml washing.Fused cell is modulated into 1 * 10 with the HAT substratum
6/ ml cultivated feeder cell 1 day in advance, and every hole 1 adds 100u in the 96 porocyte culture plates, HAT RPMI1640 cultivate 15 days (37 ℃, 5%CO
2), change HT RPMI1640 and cultivated 10 days; Step 3., get supernatant and make colony screening, to be used for the 96 hole microwell plates HER2/neu extracellular region bag quilt of screening and cloning, every hole adds the 100ul cells and supernatant, and 37 ℃ of cultivations are spent the night, the mouse-anti human IgG Fc mAb that adds the HRP mark after the washing, cultivated 30 minutes for 37 ℃, add substrate (TMB) colour developing after the washing, the OD value is positive greater than 10 * blank OD person, the result obtains the positive gram of anti-HER2/neu extracellular region .1 strain, and standard limiting dilution assay clone obtains single clone 3C19; 4. step is adjusted to 1 * 10 with positive colony 3C19 hybridoma
8/ ml gets 20ml and is expelled to SCID mouse abdominal cavity, and every 1ml got ascites after 10 days, and surveying antibody titers is 1: 16000.200 * g is centrifugal, gets supernatant, is total to 55ml, 4 ℃ of preservations; 5. step is coupled to Sephrose 4B with Protein G (Protein G) through CNBr and prepares Protein G affinity column.Protein G affinity column 0.2M glycine, pH2.3,0.1mM α-mercaptothanol BufferC pre-equilibration, and stream is washed 200ml, flow velocity 0.5ml/min.55ml contains the supernatant of total man HER2/neu monoclonal antibody, adds in the affinity column of 100ml column volume, and 4 ℃ are spent the night.Use 0.01M PB, pH8.0,0.1mM α-mercaptothanol Buffer D fully stream washes OD280 less than 0.02, flow velocity 1ml/min uses the 0.1M citrate buffer solution, pH3.0,0.1mM α-mercaptothanol Buffer H wash-out IgG, the part of collection OD280＞0.02, lyophilize, obtain the total man HER2/neu monoclonal antibody 3C19 2.34mg of affinity purification ,-20 ℃ of preservations.
Claims (7)
1, a kind of people's endothelial growth factor receptor 2 albumen total man monoclonal antibodies, it is characterized in that: it is made by following preparation method, may further comprise the steps successively,
1. cultivate HER2 sensitization human B cell;
2. the human B cell of HER2 sensitization and people's osteoma cytogamy, specific operation process is as follows: at first will
Be used to merge 1～2 * 10
6Karpas 707H and step 1. resulting 3～7 * 10
6Sensitization people
The B cytomixis dropwise added the freshly prepared PEG-2000 of 1～2ml in 2 minutes; Quick then
Splash into RPMI1640 50～60ml, centrifugal 5 minutes of 200 * g abandons supernatant, repeats to add
RPMI1640 50～60ml washing; At last fused cell is modulated into 1～2 * 10
6/ ml, the pre-cultivation
Cell, every hole adds convention amount in Tissue Culture Plate, and HAT RPMI1640 cultivates 12～15
My god, change HT RPMI1640 and cultivated 7～12 days;
The screening of 3. anti-HER2 extracellular fragment monoclonal antibody;
The amplification of 4. anti-HER2 extracellular fragment monoclonal antibody.
2, a kind of people's endothelial growth factor receptor 2 albumen total man MONOCLONAL ANTIBODIES SPECIFIC FOR methods comprise with next step:
1. cultivate HER2 sensitization human B cell;
2. the human B cell of HER2 sensitization and people's osteoma cytogamy, specific operation process is as follows: at first will
Be used to merge 1～2 * 10
6Karpas 707H and step 1. resulting 3～7 * 10
6Sensitization people
The B cytomixis dropwise added the freshly prepared PEG-2000 of 2ml in 2 minutes; Drip fast then
Go into RPMI1640 50～60ml, centrifugal 5 minutes of 200 * g abandons supernatant, repeats to add RPMI1640
50～60ml washing; At last fused cell is modulated into 1～2 * 10
6/ ml cultivated feeder cell 1 day in advance,
Every hole adds 100ul in Tissue Culture Plate, and HAT RPMI1640 cultivated 12～15 days, changes HT
RMI1640 cultivated 7～12 days;
The screening of 3. anti-HER2 extracellular fragment monoclonal antibody;
The amplification of 4. anti-HER2 extracellular fragment monoclonal antibody.
3, people's endothelial growth factor receptor 2 albumen total man MONOCLONAL ANTIBODIES SPECIFIC FOR methods as claimed in claim 2, it is characterized in that: described step 1., can realize by three approach, article one, approach is directly to extract the human B cell of HER2 sensitization from patient with breast cancer's blood of high expression level HER2/neu, at first determine the target volunteer, get patient's peripheral blood 2～5ml, measure Anti-HER 2, select its titre greater than 1: 64, take out peripheral blood 50～100ml from the target volunteer then, add B cellular segregation liquid, 200～250 * g is centrifugal, gets middle level oyster white B cellular layer; The second approach is at first to get high expression level HER2/neu patient with breast cancer's cancer week lymphoglandula, aseptic technique shreds, tryptic digestion 8～12 hours, cross 150～200 order stainless steel sifts, be suspended on the 20ml lymphocyte layering liquid, 200～250 * g is centrifugal, collects interface oyster white lymphocyte, and the bone-marrow-derived lymphocyte that will obtain in order to last method is tested with immunobead (anti-human IgG antibody of mark and anti-human IgM antibody) and whether confirmed sensitization then; Article three, approach is external HER2 immunity, with the human peripheral blood B cell of last sensitization after conditioned medium is cultivated 24 hours, change in the perfect medium and cultivated 5～7 days, the composition of described perfect medium contains HER2/neu extracellular region 1～2ng/ml, AB type human serum 1～10%, IL-2 100～150U/ml, IL-4 8～12ng/ml, IL-6 16～20ng/ml, scavenger cell 1～2 * 10
5/ ml.
4, as claim 2 or 3 described people's endothelial growth factor receptor 2 albumen total man MONOCLONAL ANTIBODIES SPECIFIC FOR methods, it is characterized in that: described step 3., the microwell plate HER2/neu extracellular region bag quilt that at first will be used for screening and cloning, every hole adds the convention amount cells and supernatant, and 37 ℃ of cultivations are spent the night; The mouse-anti human IgG Fc mAb that adds the HRP mark then after the washing cultivated 30～45 minutes for 37 ℃, added substrate TMB colour developing after the washing, and the OD value is positive greater than 10 * blank OD person.
5, people's endothelial growth factor receptor 2 albumen total man MONOCLONAL ANTIBODIES SPECIFIC FOR methods as claimed in claim 4, it is characterized in that: 4. described step is that the positive colony hybridoma is adjusted to 1～2 * 10
6/ ml gets 1ml and is expelled to nude mice abdominal cavity or SCID mouse peritoneal, gets ascites after 7～10 days, and 200～250 * g is centrifugal, gets supernatant, adds 30～50%glycerol, preserves below-20 ℃; Or the positive colony hybridoma is adjusted to 1 * 10
6/ ml gets the 20ml aseptic technique and adds in the bio-reactor, cultivates about 2 weeks, and according to the antibody titers of cell treatment system situation and sampling and measuring, it is centrifugal to get nutrient solution 200～250 * g, gets supernatant, adds 30～50% glycerol, preserves below-20 ℃.
6, people's endothelial growth factor receptor 2 albumen total man MONOCLONAL ANTIBODIES SPECIFIC FOR methods as claimed in claim 5 is characterized in that: also comprise 5. purification step of step, what this purification step adopted is affine layer suction method.
7, people's endothelial growth factor receptor 2 albumen total man MONOCLONAL ANTIBODIES SPECIFIC FOR methods as claimed in claim 6, it is characterized in that: 5. described step is people HER2/neu antigen extracellular fragment to be coupled to Sephrose 4B through CNBr prepare total man HER2/neu monoclonal antibody affinity chromatography post, described total man HER2/neu monoclonal antibody affinity chromatography post 0.01M PB, pH 6.8,0.1mM the Buffer A pre-equilibration of α-mercaptothanol, 4 ℃ of preservations, its operation is the supernatant that at first 100ml is contained total man HER2/neu monoclonal antibody, add in the affinity column of 200ml column volume, 4 ℃ are spent the night, then with Buffer A fully stream wash OD280 less than 0.01, flow velocity is 8ml/min, use 0.01M PB, pH 6.8,0.1mM α-mercaptothanol, 150mM NaCl Buffer B wash-out, collect the part of OD280＞0.02 at last, lyophilize ,-20 ℃ of preservations; Or be coupled to Sephrose 4B with albumin A (SPA) through CNBr and prepare the SPA affinity column.SPA affinity column 0.2M glycine, pH 2.3,0.1mM α-mercaptothanol Buffer C pre-equilibration, and stream is washed 200ml, flow velocity 0.5ml/min, its operation is the supernatant that at first 100ml is contained total man HER2/neu monoclonal antibody, adds in the affinity column of 100ml column volume, and 4 ℃ are spent the night, use 0.01M PB then, pH 8.0, and 0.1mM α-mercaptothanol Buffer D fully stream washes OD280 less than 0.02, flow velocity 1ml/min, use the 0.1M citrate buffer solution, pH6.0,0.1mM α-mercaptothanol Buffer E wash-out IgG1 uses the 0.1M citrate buffer solution, pH3.5,0.1mM α-mercaptothanol Buffer F wash-out IgG2b uses the 0.1M citrate buffer solution, pH4.4,0.1mM α-mercaptothanol Buffer G wash-out IgG2a, collect the part of OD280＞0.02 at last, lyophilize ,-20 ℃ of preservations; Or be coupled to Sephrose 4B with Protein G (Protein G) CNBr and prepare Protein G affinity column, Protein G affinity column 0.2M glycine, pH 2.3,0.1mM α-mercaptothanol Buffer C pre-equilibration, and stream is washed 200ml, flow velocity 0.5ml/min, its operation is the supernatant that at first 100ml is contained total man HER2/neu monoclonal antibody, adds in the affinity column of 100ml column volume, and 4 ℃ are spent the night, use 0.01M PB then, pH 8.0, and 0.1mM α-mercaptothanol BufferD fully stream washes OD280 less than 0.02, flow velocity 1ml/min, use the 0.1M citrate buffer solution, pH3.0,0.1mM α-mercaptothanol Buffer H wash-out IgG collects the part of OD280＞0.02 at last, lyophilize ,-20 ℃ of preservations.
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