CN102241773B - Anti-myeloma cell polyclonal antibody and preparation method thereof - Google Patents
Anti-myeloma cell polyclonal antibody and preparation method thereof Download PDFInfo
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- CN102241773B CN102241773B CN201010171796.9A CN201010171796A CN102241773B CN 102241773 B CN102241773 B CN 102241773B CN 201010171796 A CN201010171796 A CN 201010171796A CN 102241773 B CN102241773 B CN 102241773B
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Abstract
The invention which belongs to the field of antibody preparation and application concretely relates to an anti-myeloma cell polyclonal antibody, a preparation method thereof and an application thereof. The myeloma cell polyclonal antibody of the invention which is prepared from myeloma cell immune animals can be used in preparing reagents for detecting or diagnosing multiple myeloma and medicaments for treating multiple myeloma. The myeloma cell polyclonal antibody of the present invention, which is a polyclonal antibody prepared through treating whole myeloma cells as an antigen, can simultaneously direct to multiple antigens on myeloma cell surfaces, and is in favor of enhancing the treatment effect on clinical myeloma patients.
Description
Technical field
The invention belongs to antibody preparation and application field.Be specifically related to polyclonal antibody of a kind of anti-myeloma cell and preparation method thereof and purposes.
Background technology
Multiple myeloma (multiple myeloma, MM) is the second largest common cancer of blood system, accounts for 10% of hematologic malignancies, accounts for 1% of human malignancies.The annual morbidity of China approximately 1,/10 ten thousand people.Along with the aging of population, the sickness rate of China's multiple myeloma also constantly rises.Traditional chemicotherapy, autologous stem cell transplantation and heteroplastic transplantation is all difficult to cure myelomatosis.Therefore be badly in need of the new medicine of exploitation and seek new treatment approach.
Along with various tumor associated antigens are out identified myeloma cell, as CD20, CD40, CD52, CD33, CD138, MUC1, HM1.24, CYP1B1, SP17, PRAME, Wilms ' tumour 1 (WT1) and gp96.The monoclonal antibody of target surface of myeloma cells specific antigens has been given play to important effect in the diagnosis of multiple myeloma and treatment.The go on the market anti-CD-20 monoclonal antibody (commodity are called Mabthera, Rituximab) of (being ratified by U.S. FDA for 1997), effect of its treatment myelomatosis is remarkable.In addition, multiple monoclonal antibody comprises that SGN-40 (anti-huCD40), anti-CD126 (atlezumab), anti-CD52 (alemtuzumab), MRA (humanizedanti-interleukin-6 (IL-6) antibody, atlizumab), TRM-1 (Fully Human TRAIL-R1MoAb), AHM (anti-HM1.24MoAb) have also entered clinical experimental stage.But, due to myeloma cell's heterogeneity, and the limitation of monoclonal antibody target spot, causing in the time for the treatment of multiple myeloma, the effect of monoclonal antibody is limited sometimes.Such as, CD20 only detects and has expression in 13～22% patient.Therefore, not all patient is using the reaction that can produce expection after existing antibody drug.So exploitation has the medicine of specificity and broad spectrum by the important development direction that is myelomatosis treatment field for tumour cell.
Compared with monoclonal antibody, polyclonal antibody may be a kind of selection, because its multiple antigen of target simultaneously, thereby may trigger different necrocytosis approach simultaneously.In addition, polyclonal antibody also can reach sufficiently high density at tumor cell surface, be conducive to Fc acceptor or the C1q on effector cell surface crosslinked mutually, thereby trigger cytotoxic effect (the antibody-dependent cell-mediated cytotoxicity of antibody-dependant cell mediation, and the cytotoxicity of Complement Dependent (Complementdependent cytotoxicity, CDC) effect ADCC).And Anti-TNF-α physical efficiency is minimally avoided the appearance of the tumour cell mutant occurring due to drug resistance, very low because tumour cell is lost the probability of its surface antigen simultaneously.
Although, also there is at present investigator to attempt treating myelomatosis with polyclonal antibody, but that adopt is the thin immunoglobulin (Ig) of anti-thymus gland (Polyclonal antithymocyte globulins, and antiangiogenic thin immunoglobulin (Ig) (Polyclonal antilymphocyte globulins, ALG) ATG).At present, have commercialization ATG and ALG, be mainly used in the treatment of organ transplantation immunological rejection.The preparation of ATG or ALG is mainly using T cell or lymphocyte as antigen immune rabbit or horse and obtaining, and therefore, its identification myeloma cell ability may be not enough, in clinical application, may act on limited to the patient of high myelomatosis burden.So, to develop more effectively, the polyclonal antibody of high recognition capability and wide spectrum has clear and clear and definite medical need.
Summary of the invention
The object of this invention is to provide polyclonal antibody of a kind of full cell surface specific antigens of myelomatosis for living and preparation method thereof and purposes.
Myeloma cell's polyclonal antibody provided by the invention is with making after myeloma cell immune animal,
Further, myeloma cell's polyclonal antibody of the present invention is to be made by following methods:
A, use 1 × 10 first
6～5 × 10
7the myeloma cell of individual work and complete Freund's adjuvant immune animal;
B, then every 7～14 days booster immunizations once, booster immunization too many or too much for use full freund's adjuvant and myeloma cell's mixed immunity of living; Before each immunity, all detect blood antibody titers, when antibody titers reaches 1: 10000～more than 50000 termination immunity;
C, spend the night in 4 degrees Celsius after stopping getting blood after immunity place 2～3h under room temperature, then get the centrifugal serum obtaining containing myeloma cell's polyclonal antibody of supernatant, from making myeloma cell's polyclonal antibody containing purifying the serum of myeloma cell's polyclonal antibody.
Wherein, in aforesaid method step c, from the method that makes myeloma cell's polyclonal antibody containing purifying the serum of myeloma cell's polyclonal antibody be:
A, will be splined on the pillar by the buffer A balance of 10 times of column volumes containing the serum of myeloma cell's polyclonal antibody, pillar filler is Mabselect, then washes pillar by the buffer A of 5 times of column volumes; Described buffer A is 20mM NaH
2pO
4, 0.15M NaCl, pH 7.2;
B, make buffer B linear gradient with the 0.1M Trisodium Citrate of pH 3.0 and wash pillar, until there is the appearance of elution peak;
C, collection elution peak liquid do neutralization buffer with the Tris-HCl of pH8.0,1M simultaneously, regulate elutriant to pH7.2 left and right;
D, then the elutriant of collection is placed in to PH7.2 with dialysis tubing PBS damping fluid in 4 lower dialysis Celsius, the collection liquid freeze-drying of dialysis is obtained to myeloma cell's polyclonal antibody.
The present invention also provides the purposes of above-mentioned myeloma cell's polyclonal antibody in the reagent of preparation detection or diagnose multiple myeloma.
The present invention also provides the purposes of above-mentioned myeloma cell's polyclonal antibody in the medicine of preparation treatment multiple myeloma.
The present invention also provides the method for the above-mentioned myeloma cell's polyclonal antibody of purifying, and the method is:
Serum containing myeloma cell's polyclonal antibody is splined on to Buffer A (the 20mM NaH with 10 times of column volumes
2pO
4, 0.15M NaCl, pH 7.2 pH 9.0) and the XK16/40 pillar of balance, filler is Mabselect, then washes pillar with the Buffer A of 5 times of column volumes;
Wash post by Buffer B (0.1M citric acid is received, pH 3.0) linear gradient, until there is the appearance of elution peak;
Collect elution peak liquid, in simultaneously using and Buffer (1M Tris-HCl, pH8.0) adjusting elutriant pH7.2 left and right;
Then the elutriant of collection is placed in to PBS damping fluid (PH7.2) with dialysis tubing and dialyses under 4 degrees Celsius, the collection liquid freeze-drying of dialysis is obtained to myeloma cell's polyclonal antibody.
The present invention also provides the method for preparing above-mentioned myeloma cell's polyclonal antibody, and the method comprises the following steps:
A, use 1 × 10 first
6～5 × 10
7the myeloma cell of individual work and complete Freund's adjuvant immune animal;
B, then every 7～14 days booster immunizations once, booster immunization too many or too much for use full freund's adjuvant and myeloma cell's mixed immunity of living; Before each immunity, all detect serum antibody titer, when antibody titers reaches 1: 10000～more than 50000 termination immunity;
C, spend the night in 4 degrees Celsius after stopping getting blood after immunity place 2～3h under room temperature, then get the centrifugal serum obtaining containing myeloma cell's polyclonal antibody of supernatant, make myeloma cell's polyclonal antibody from serum purifying.
Further described in aforesaid method, from purge process be:
Serum containing myeloma cell's polyclonal antibody is splined on to Buffer A (the 20mM NaH with 10 times of column volumes
2pO
4, 0.15M NaCl, pH 7.2pH 9.0) and the XK16/40 pillar of balance, filler is Mabselect;
Wash pillar with the Buffer A of 5 times of column volumes again; Wash post by Buffer B (0.1M sodium citrate, pH 3.0) linear gradient, until there is the appearance of elution peak; Collect elution peak liquid, in simultaneously using and Buffer (1M Tris-HCl, pH8.0) adjusting elutriant pH7.2 left and right;
The elutriant of collection is placed in to PBS damping fluid (PH7.2) with dialysis tubing and under 4 degrees Celsius, dialyses, the collection liquid freeze-drying of dialysis is obtained to myeloma cell's polyclonal antibody.
The above method of the present invention is suitable for various mouse or human myeloma cell and extracts after serum and prepare polyclonal antibody by affinity chromatography as antigen-immunized animal.Myeloma cell line used can be mouse MPC-11 or people ARH-77 cell, or other human myeloma cell line (as U266, J41MT, ARP-1, OPM1, OPM-2, KM3, RPMI8226 etc.), or the target animal of the myeloma cell who separates from clinical patients with malignant myeloma peripheral blood, immunity can be this area Dispersal risk various conventional animals used, as rabbit, horse, ox etc.
The invention has the beneficial effects as follows: due to the polyclonal antibody that the present invention be directed to full surface of myeloma cells antigen alive and prepare, thereby guarantee specificity and broad spectrum to myeloma cell, thereby be conducive to the detection to potential ill risk and the treatment to patients with malignant myeloma.And, also the new treatment target spot of myeloma cell is explored and laid a good foundation.
Accompanying drawing explanation
Fig. 1. be that the present invention is for ELISA result photo.
Fig. 2. be the photo of the present invention for immunoblotting.Loading is mouse myeloma cell line MPC-11, and SP2/0 and NS-1 cell whole protein can find from figure, antibody of the present invention, except can effectively identifying MPC-11 albumen, can also be identified other mouse myeloma cell lines SP2/0 and NS-1.
Fig. 3. be that the present invention is for MPC-11 cellular immunofluorescence Photomicrograph.
Fig. 4. be that the present invention is for mouse myeloma cell line immunofluorescence cell streaming photo.Upper row's mouse myeloma cell line, from left to right: MPC-11, SP2/0, NS-1, lower row people source myeloma cell line, from left to right: Raji, U266, ARH-77.
Fig. 5. be the belly cavity tumor model of polyclonal antibody treatment, from figure, we can find out that polyclonal antibody treatment group obviously suppresses the growth of MCP-11.
Fig. 6. be polyclonal antibody treatment group, the heart of control antibodies group and physiological saline group mouse, liver, spleen, the HE photo of lung and kidney, from figure, we can find out, give polyclonal antibody group mouse compared with control group mice, main organs does not have obvious pathological change.
Fig. 7. be polyclonal antibody treatment group, the heart of control antibodies group and physiological saline group mouse, liver, spleen, the HE photo of lung and kidney, from figure, we can find out, give polyclonal antibody group mouse compared with control group mice, main organs does not have obvious pathological change.
Fig. 8. being polyclonal antibody analyzes the MTT of normal mouse boosting cell, and from figure, we can find out that polyclonal antibody is basically identical for the splenocyte effect of normal mouse and physiological saline and control antibodies treatment effect, do not show any cytotoxic effect.
Embodiment
Below in conjunction with accompanying drawing, by embodiment, the present invention is further elaborated.
Clone and animal
Myeloma cell line (mouse MPC-11 or people ARH-77) is purchased from U.S. ATCC (American TyPe Culture Collection).
6-8 age in week, female BALB/c mouse and new zealand white rabbit came from Sichuan University's West China Experimental Animal Center;
6-8 age in week, female SCID mouse came from Nanjing University's model animal center.
Main agents
Mabselect filler: purchased from U.S. GE (general) company.
Hoechest dyestuff: purchased from German Hearst (Hoechst) company.
Poly-lysine: purchased from U.S. Sigma (Sigma) company.
Protein molecular weight standard product: purchased from Canadian MBI Fermentas (rich enzyme safe this) company.
Quantification of protein reagent (protein assay) is purchased from U.S. Bio-Rad (Bole) company.
Poly(vinylidene fluoride) pvdf membrane: purchased from Switzerland Roche (Roche Holding Ag) company;
RPMI1640 substratum tire, bovine serum (FCS): purchased from Gibico BRL company of the U.S.;
MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 3-(4,5) dimethylbenzene thiazole-2,5 biphenyl Thiazolyl blue tetrazolium bromides, purchased from U.S. Sigma (Sigma) company;
The goat anti-rabbit igg of horseradish peroxidase-labeled: purchased from Beijing Zhong Shan Bioisystech Co., Ltd, import packing;
FITC mark goat anti-rabbit igg: purchased from Beijing Zhong Shan Bioisystech Co., Ltd, import packing;
DAB colouring reagents box: purchased from Wuhan Boster Biological Technology Co., Ltd.;
Other reagent are import or domestic analytical pure product.
Experimental instruments
Low speed centrifuge: Megaufgel.0, purchased from German Heraeus (congratulating Li Shi) company;
Supercentrifuge: eentriufge5415C, purchased from German Eppendorf (Ai Bende) company;
High speed low temperature centrifugal machine: Biofuge28RS, purchased from German Heraeus (congratulating Li Shi) company;
Ultralow Temperature Freezer (80 ℃): purchased from Japanese Sanyo (Sanyo) company;
Constant incubator: purchased from Japanese Sanyo (Sanyo) company;
Pure water instrument: EASYPure UF07412, purchased from Millipore company (Mi Libo, the U.S.);
High-pressure sterilizing pot: LaboAut ℃ lave, purchased from Sanyo company (Sanyo, Japan);
Vertical electrophoresis system: MiniPROTEAN2 & 3Cell, purchased from U.S. Bio-Rad (Bole) company;
Inverted microscope: Lx50 Olympus, purchased from Japanese Olympus (Olympus) company;
Protein purification system:
explorer, purchased from U.S. GE (general) company.
Embodiment mono-. the Preparation and identification of the anti-mouse of rabbit or the full myeloma cell's of people polyclonal antibody
1, the preparation of the anti-mouse of rabbit or the full myeloma cell's of people polyclonal antibody
Use the full cell of myelomatosis (mouse MPC-11 or people ARH-77) and complete Freund's adjuvant immunity new zealand white rabbit of living, after 7～14 days, get blood 1ml from the auricular vein of new zealand white rabbit, solidify rear centrifuging and taking serum, ELISA method detects the titre of polyclonal antibody, it is 1: 2000, after the full freund's adjuvant that tood many or too much for use every 7～14 days mix booster immunization with the full myeloma cell who lives, before each immunity, all get blood 1ml from auricular vein, ELISA method detects antibody titers.When antibody titers reach 1: 10000～more than 1: 50000, get blood 80m from every new zealand white rabbit carotid artery and be contained in glass triangle flask (250ml), under room temperature, place 2～3h, then 4 degrees Celsius of refrigerator overnight.Next day, draw the supernatant in Erlenmeyer flask, 2000 centrifugal 10 minutes, get supernatant, at inferior 10000rpm centrifugal 10 minutes, get supernatant and carry out antibody purification, or-80 degrees Celsius are frozen to antibody purification.
The purifying of IgG antibody carries out according to Mabselect specification sheets.In brief, pillar is chosen XK16/40, and filler is selected Mabselect.Purification system uses
explorer system.In conjunction with Buffer A (20mM NaH
2pO
4, 0.15M NaCl, pH 7.2) and the packing of centrifugal collection antibody serum, adopt Mabselect purified rabbit Immunoglobulin in Serum IgG, Bio-Rad method is measured antibody concentration and is reached 20mg/ml, Freeze Drying Equipment freeze-drying after packing, and preserve-80 ℃.
Concrete purification process is:
With Buffer A (the 20mM NaH of 10 times of pillar volumes
2pO
4, 0.15M NaCl, pH 7.2 pH 9.0) and balance XK16/40 pillar;
Loading immunize rabbit serum or contrast blank rabbit anteserum (50ml);
Wash pillar with the BufferA of 5 times of column volumes again;
Wash post with linear gradient Buffer B (0.1M citric acid is received, pH 3.0), until there is the appearance of elution peak;
Collect elution peak liquid, in then using immediately and Buffer (1M Tris-HCl, pH8.0) adjusting eluting liquid pH7.2 left and right.
Then use dialysis tubing as for PBS (PH7.2) dialysed overnight in 4 degrees Celsius of chromatography cabinets the elutriant of collecting.
The collection liquid of dialysed overnight is obtained to the IgG powder of purifying with refrigerator freeze-drying ,-80 degrees Celsius of Refrigerator stores.
The preparation that above method is suitable for various mouse or human myeloma cell extracts polyclonal antibody as antigen-immunized animal especially rabbit affinity chromatography.
2, the evaluation of the anti-mouse of rabbit or the full myeloma cell's of people polyclonal antibody
1) cell ELISA: myeloma cell is layered in 96 coated orifice plates of poly-lysine by 10000 amount, and not immune rabbit igg and polyclonal antibody (immunize rabbit IaG) are pressed 1: 2000,1: 5000,1: 10000, make an antibody gradient, be ELISA at 1: 20000; (Fig. 1)
The results are shown in Figure 1, result shows that antibody group of the present invention in conjunction with myeloma cell's ability is 8～10 times of control antibodies binding ability, and has dose-dependent effect, along with the increase antibody group of Dilution ratio decreases in conjunction with myelomatosis ability.
2) immunoblotting: collect 10
6cell pyrolysis liquid is pressed different concns loading electrophoresis, transferring film, 5% skim-milk is as confining liquid, room temperature closed protein electricity transferring film 1 hour, the anti-human full myeloma cell's Anti-TNF-α bulk concentration of rabbit be 5 μ g/ μ l as primary antibodie, 1: 1000～1: 5000 dilution, incubated at room 2 hours or 4 ℃ of overnight incubation, 1 × TBST damping fluid will be washed 3 times, each 5 minutes; The goat anti-rabbit igg of horseradish peroxidase-labeled is anti-as two, dilution in 1: 5000, and incubated at room 60 minutes, 1 × TBST damping fluid shakes washes 3 times, and each 5～15 minutes, the colour developing of horseradish peroxidase substrate, compressing tablet punching exposure.
The results are shown in Figure 2, result shows that polyclonal antibody can effectively identify different multiple myeloma cell lines, but show the different myeloma cell line ability differences of identification (from left to right: MPC-11, SP2/0, NS-1) of polyclonal antibody from protein band.
3) immunofluorescent staining: by 10
6cell is fixed with 100 μ l4% paraformaldehydes, then on the slide glass that smear was processed in poly-lysine, 10% lowlenthal serum room temperature closing cell 1 hour, the anti-human full myeloma cell's Anti-TNF-α bulk concentration of rabbit is 5 μ g/ μ l dilutions, room temperature reaction 30～60 minutes, 1 × PBS shakes and washes 3 times, each 5～15 minutes; With two dilutions in anti-1: 500 of the green fluorescence goat-anti rabbit of FITC mark, room temperature reaction 30～60 minutes, two anti-sealings finish first 5 minutes, and hoechest dyes core, and 1 × PBS shakes and washes 3 times, each 5～15 minutes; Mounting Microscopic observation; (Fig. 3)
The results are shown in Figure 3, result shows, uses antibody group of the present invention, can see green fluorescence at MPC-11 cell surface, and contrast normal rabbit serum immunoglobulin (Ig) fails to see green fluorescence.Therefore antibody of the present invention can specific recognition MPC-11 cell-surface antigens, and contrast normal rabbit serum immunoglobulin (Ig) can not be identified.
4) cell streaming: by the myeloma cell of logarithmic phase 10
6by the anti-human full myeloma cell's polyclonal antibody 5 μ g/ μ l incubated at room of rabbit 30～60 minutes, 1 × PBS shook and washes 3 times, each 5～15 minutes; With two dilutions in anti-1: 500 of the green fluorescence goat-anti rabbit of FITC mark, room temperature reaction 30～60 minutes, two anti-sealings finish to shake and wash 3 times with 1 × PBS, and each 5～15 minutes, at least 10000 cell upflowing cell instruments detections.(Fig. 4)
The results are shown in Figure 4, gray line represent mouse of the present invention source or human antibody group respectively with the reaction of corresponding mouse source or people source multiple myeloma cells, black line represents that control antibodies reacts as negative control with mouse source or people source multiple myeloma cells respectively, result shows, antibody group fluorescence intensity of the present invention all oriented right avertence is moved, and proves that antibody group of the present invention can effectively identify multiple multiple myeloma cells.(above arrange mouse myeloma cell line, from left to right: MPC-11, SP2/0, NS-1, lower row people source myeloma cell line, from left to right: Raji, U266, ARH-77)
Tumor killing effect in the body of the polyclonal antibody of embodiment bis-anti-myeloma cells
First by 21 Babl/c mouse peritoneal inoculations 10
6the MPC-11 cell of left and right, is divided into three groups, blank group (PBS) after 5 days; Control antibodies group (non-immune rabbit igg); Experimental group (rabbit igg of immunity is polyclonal antibody group), 7 every group.Then distinguish abdominal injection PBS, not immune rabbit igg (200 μ g/ml) or polyclonal antibody (200 μ g/ml), every other day injection once, has altogether six times.After the 6th injection, within second day, put to death mouse, intraperitoneal tumor nodule number is calculated in necrotomy.(Fig. 5)
The results are shown in Figure 5, control antibodies and physiological saline group mouse peritoneal tubercle number are obviously more than antibody group mouse peritoneal tubercle number of the present invention, and abdominal cavity tubercle number is larger, magnitude range is in 0.2～1.5cm left and right, and antibody group mouse peritoneal tubercle magnitude range of the present invention is in about 0.1～0.6cm, result shows that antibody of the present invention has the effect of obvious inhibition multiple myeloma at mouse tumor growth.
Embodiment tri-. tumor killing effect in the body of the polyclonal antibody of xenotransplantation anti-myeloma cell
First by 21 right back of the body subcutaneous abdomen inoculations 10 of SCID mouse
6the people source multiple myeloma cells ARH-77 of left and right, when laying one's hand on and it is divided into three groups at random, blank group (NS) when tumor nodule; Control antibodies group (non-immune rabbit igg); Experimental group (rabbit igg of immunity is polyclonal antibody group), 7 every group.Then distinguish tail vein injection NS, not immune rabbit igg (200 μ g/ml) or polyclonal antibody (200 μ g/ml) 100 microlitres, every other day injection once, has altogether six times.In order to verify that antibody group of the present invention, to inhibition in the body of xenotransplantation tumour, uses vernier caliper measurement gross tumor volume, every other day measure once, draw tumor growth curve.
The results are shown in Figure 6, ◆, ■, ▲ representing that respectively NS, control antibodies and of the present invention group of antibody treatment mouse tumor growth volume, result show, antibody group of the present invention is processed mouse can obviously suppress tumour tumor growth.
The polyclonal antibody safety evaluation of embodiment tetra-anti-myeloma cells of the present invention
Get and accept polyclonal antibody treatment (200 μ g/ml) group, with the heart of mouse that gives control antibodies group and physiological saline group, liver, spleen, lung and nephridial tissue, carry out HE dyeing, observe give polyclonal antibody treatment after main organs whether have damage.Further by using MTT method
The results are shown in Figure 7, result shows antibody group mouse core of the present invention, liver, and spleen, lung is compared with physiological saline group with control antibodies group with nephridial tissue, does not have obvious pathological characters and changes.
Get normal mouse splenocyte, the control antibodies and the polyclonal antibody (200g/ml) that give respectively physiological saline, same concentrations are processed, and after 48 hours, analyze by MTT, observe polyclonal antibody and process the cytotoxicity to normal mouse boosting cell.
The results are shown in Figure 8, the splenocyte effect of antibody of the present invention to normal mouse and physiological saline and control antibodies treatment effect are basically identical, do not show any cytotoxic effect.
Claims (2)
1. the purposes of myeloma cell's polyclonal antibody in the medicine of preparation treatment multiple myeloma, described myeloma cell's polyclonal antibody is to be made by following methods:
A, use 1 × 10 first
6～5 × 10
7the full cell of myelomatosis and the complete Freund's adjuvant immune animal of individual work, described animal is rabbit;
B, then every 7～14 days booster immunizations once, booster immunization too many or too much for use full freund's adjuvant and the full cytomixis immunity of myelomatosis of living; Before each immunity, all detect blood antibody titers, stop above immunity when antibody titers reaches 1:10000;
C, spend the night in 4 degrees Celsius after stopping getting blood after immunity place 2～3h under room temperature, then get the centrifugal serum obtaining containing myeloma cell's polyclonal antibody of supernatant, from making myeloma cell's polyclonal antibody containing purifying the serum of myeloma cell's polyclonal antibody.
2. purposes according to claim 1, is characterized in that, the step of described purifying is:
Serum containing myeloma cell's polyclonal antibody is splined on to the XK16/40 post by the buffer A balance of 10 times of column volumes, and filler is Mabselect;
Wash pillar by the buffer A of 5 times of column volumes again; Wash post by buffer B linear gradient, until there is the appearance of elution peak; Collect elution peak liquid, regulate elutriant pH7.2 by neutralization buffer simultaneously;
The elutriant of collection is dialysed under 4 degrees Celsius with the PBS damping fluid that dialysis tubing is placed in pH7.2, the collection liquid freeze-drying of dialysis is obtained to myeloma cell's polyclonal antibody;
Above-mentioned buffer A is 20mM NaH
2pO
4, 0.15M NaCl, pH7.2; Buffer B is 0.1M Trisodium Citrate, pH3.0; Neutralization buffer is 1M Tris-HCl, pH8.0.
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