CN102241773A - 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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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 Areas.Be specifically related to a kind of polyclonal antibody and preparation method thereof and purposes of anti-myeloma cell.
Background technology
(multiple myeloma is the second largest common cancer of blood system MM) to multiple myeloma, accounts for 10% of hematologic malignancies, accounts for 1% of human malignancies.About 1,/10 ten thousand people of the annual morbidity of China.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 all are 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 identified out the 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.Go on the market (1997 by drugs approved by FDA) anti-CD-20 monoclonal antibody (commodity are called Mabthera, Rituximab), its treatment myelomatosis effect remarkable.In addition, multiple monoclonal antibody comprise 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) also entered clinical experimental stage.But, because myeloma cell's heterogeneity, and the limitation of monoclonal antibody target spot, causing when the treatment multiple myeloma, the effect of monoclonal antibody is limited sometimes.Such as, CD20 only detects in 13～22% patient expression.Therefore, not all patient can both produce the reaction of expection after using existing antibody drug.So exploitation will be the important development direction in myelomatosis treatment field at the medicine that tumour cell has specificity and broad spectrum.
Compare with monoclonal antibody, polyclonal antibody may be a kind of selection, because its a plurality of antigens of target simultaneously, thereby may trigger different necrocytosis approach simultaneously.In addition, polyclonal antibody also can reach sufficiently high density at tumor cell surface, help mutually crosslinked with the Fc acceptor or the C1q on effector cell surface, thereby trigger cytotoxic effect (the antibody-dependent cell-mediated cytotoxicity of antibody-dependant cell mediation, ADCC) and the complement cytotoxicity (Complementdependent cytotoxicity, CDC) effect that rely on.And, polyclonal antibody can minimum degree avoid the appearance of the tumour cell mutant that takes place owing to drug resistance very low because tumour cell is lost the probability of its surface antigen simultaneously.
Though, also there is the investigator to attempt treating myelomatosis at present with polyclonal antibody, but adopt be the thin immunoglobulin (Ig) of anti-thymus gland (Polyclonal antithymocyte globulins, ATG) and antiangiogenic thin immunoglobulin (Ig) (Polyclonal antilymphocyte globulins, ALG).At present, have commercialization ATG and ALG, be mainly used in the treatment of organ transplantation immunological rejection.The preparation of ATG or ALG mainly is to obtain as antigen immune rabbit or horse with T cell or lymphocyte, and therefore, its identification myeloma cell ability may be not enough, may act on limited to the patient of high myelomatosis burden in clinical application.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 purpose of this invention is to provide a kind of polyclonal antibody at the full cell surface specific antigens of myelomatosis of living and preparation method thereof and purposes.
Myeloma cell's polyclonal antibody provided by the invention is with making behind the myeloma cell immune animal,
Further, myeloma cell's polyclonal antibody of the present invention is to be made by following method:
A, use 1 * 10 first
6～5 * 10
7The myeloma cell of individual work and complete Freund's adjuvant immune animal;
B, then once, booster immunization too many or too much for use full freund's adjuvant and myeloma cell's mixed immunity of living every 7～14 days booster immunizations; All detect the blood antibody titers before each immunity, when antibody titers reaches 1: 10000～termination immunity more than 50000;
C, stop getting after the immunity blood and spend the night in 4 degrees centigrade after placing 2～3h under the room temperature, get the centrifugal serum that obtains containing myeloma cell's polyclonal antibody of supernatant then, purifying makes myeloma cell's polyclonal antibody from the serum that contains myeloma cell's polyclonal antibody.
Wherein, the method that purifying makes myeloma cell's polyclonal antibody from the serum that contains myeloma cell's polyclonal antibody among the aforesaid method step c is:
A, the serum that will contain myeloma cell's polyclonal antibody are splined on the buffer A equilibrated pillar with 10 times of column volumes, and the pillar filler is Mabselect, washes pillar with the buffer A of 5 times of column volumes again; Described buffer A is 20mM NaH
2PO
4, 0.15M NaCl, pH 7.2;
B, make the buffer B linear gradient with the 0.1M Trisodium Citrate of pH 3.0 and wash pillar, until the appearance that elution peak is arranged;
C, collect elution peak liquid, the Tris-HCl with pH8.0,1M does neutralization buffer simultaneously, and the adjusting elutriant is to the pH7.2;
D, the elutriant of collecting is placed PH7.2 with dialysis tubing then the PBS damping fluid in 4 dialysis down Celsius, the collection liquid freeze-drying of dialysis is promptly got 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 this method is:
The serum that will contain myeloma cell's polyclonal antibody is splined on Buffer A (the 20mM NaH with 10 times of column volumes
2PO
4, 0.15M NaCl, pH 7.2pH 9.0) and equilibrated XK16/40 pillar, filler is Mabselect, the BufferA with 5 times of column volumes washes pillar again;
Wash post with Buffer B (the 0.1M citric acid is received, and pH 3.0) linear gradient, until the appearance that elution peak is arranged;
Collect elution peak liquid, in using simultaneously and Buffer (1M Tris-HCl is pH8.0) about adjusting elutriant pH7.2;
Place PBS damping fluid (PH7.2) 4 degrees centigrade of dialysis down with dialysis tubing the elutriant of collecting then, the collection liquid freeze-drying of dialysing is promptly got myeloma cell's polyclonal antibody.
The present invention also provides the method for preparing above-mentioned myeloma cell's polyclonal antibody, and this method may further comprise the steps:
A, use 1 * 10 first
6～5 * 10
7The myeloma cell of individual work and complete Freund's adjuvant immune animal;
B, then once, booster immunization too many or too much for use full freund's adjuvant and myeloma cell's mixed immunity of living every 7～14 days booster immunizations; All detect serum antibody titer before each immunity, when antibody titers reaches 1: 10000～termination immunity more than 50000;
C, stop getting after the immunity blood and after placing 2～3h under the room temperature, spend the night in 4 degrees centigrade, get the centrifugal serum that obtains containing myeloma cell's polyclonal antibody of supernatant then, make myeloma cell's polyclonal antibody from the serum purifying.
Further described in the aforesaid method be from purge process:
The serum that will contain myeloma cell's polyclonal antibody is splined on Buffer A (the 20mM NaH with 10 times of column volumes
2PO
4, 0.15M NaCl, pH 7.2pH 9.0) and equilibrated XK16/40 pillar, filler is Mabselect;
Buffer A with 5 times of column volumes washes pillar again; Wash post with Buffer B (0.1M sodium citrate, pH 3.0) linear gradient, until the appearance that elution peak is arranged; Collect elution peak liquid, in using simultaneously and Buffer (1M Tris-HCl is pH8.0) about adjusting elutriant pH7.2;
Place PBS damping fluid (PH7.2) 4 degrees centigrade of dialysis down with dialysis tubing the elutriant of collecting, the collection liquid freeze-drying of dialysing is promptly got myeloma cell's polyclonal antibody.
Suitable various mouse of the above method of the present invention or human myeloma cell prepare polyclonal antibody with affinity chromatography after extracting serum as antigen-immunized animal.Used myeloma cell line can be mouse MPC-11 or people ARH-77 cell, perhaps other human myeloma cell line (as U266, J41MT, ARP-1, OPM1, OPM-2, KM3, RPMI8226 etc.), perhaps isolating myeloma cell, immune target animal can be this area and prepare the used various conventional animals of antibody from clinical myelomatosis patient's peripheral blood, as rabbit, horse, ox etc.
The invention has the beneficial effects as follows: owing to The present invention be directed to the polyclonal antibody of full surface of myeloma cells antigen prepd alive, thereby guaranteed specificity and broad spectrum, thereby helped to the detection of potential ill risk and to myelomatosis patient's treatment to the myeloma cell.And, also the new treatment target spot of myeloma cell is explored and laid a good foundation.
Description of drawings
Fig. 1. be that the present invention is used for ELISA photo as a result.
Fig. 2. be the photo that the present invention is used for immunoblotting.Last sample is mouse myeloma cell line MPC-11, and SP2/0 and NS-1 cell whole protein can find that from figure antibody of the present invention can also be discerned other mouse myeloma cell lines SP2/0 and NS-1 except can effectively discerning the MPC-11 albumen.
Fig. 3. be that the present invention is used for MPC-11 cellular immunofluorescence Photomicrograph.
Fig. 4. be that the present invention is used for mouse myeloma cell line immunofluorescence cell streaming photo.Last row's mouse myeloma cell line, from left to right: MPC-11, SP2/0, NS-1, following 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 as can be seen polyclonal antibody treatment group obviously suppress 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, we are as can be seen from figure, give polyclonal antibody group mouse and compare with control group mice, main organs does not have tangible 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, we are as can be seen from figure, give polyclonal antibody group mouse and compare with control group mice, main organs does not have tangible pathological change.
Fig. 8. being polyclonal antibody analyzes the MTT of normal mouse boosting cell, from figure we as can be seen polyclonal antibody do not show any cytotoxic effect for splenocyte effect and the physiological saline and the control antibodies treatment effect basically identical of normal mouse.
Embodiment
The present invention is further elaborated by embodiment below in conjunction with accompanying drawing.
Clone and animal
Myeloma cell line (mouse MPC-11 or people ARH-77) is purchased in U.S. ATCC (American TyPe CultureCollection).
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 model animal center.
Main agents
Mabselect filler: available from U.S. GE (general) company.
Hoechest dyestuff: available from German Hearst (Hoechst) company.
Poly-lysine: available from U.S. Sigma (Sigma) company.
Protein molecular weight standard product: available from Canadian MBI Fermentas (rich enzyme safe this) company.
Quantification of protein reagent (protein assay) is available from U.S. Bio-Rad (Bole) company.
Poly(vinylidene fluoride) pvdf membrane: available from Switzerland Roche (Roche Holding Ag) company;
RPMI1640 substratum tire, bovine serum (FCS): available from U.S. Gibico BRL company;
MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazol ium bromide, 3-(4,5) dimethylbenzene thiazole-2,5 biphenyl Thiazolyl blue tetrazolium bromide is available from U.S. Sigma (Sigma) company;
The goat anti-rabbit igg of horseradish peroxidase-labeled: available from Beijing Zhong Shan Bioisystech Co., Ltd, import packing;
FITC mark goat anti-rabbit igg: available from Beijing Zhong Shan Bioisystech Co., Ltd, import packing;
DAB colouring reagents box: available from Wuhan Boster Biological Technology Co., Ltd.;
Other reagent are import or homemade analytical pure product.
Laboratory apparatus and equipment
Low speed centrifuge: Megaufgel.0 is available from German Heraeus (congratulating Li Shi) company;
Supercentrifuge: eentriufge5415C is available from German Eppendorf (Ai Bende) company;
High speed low temperature centrifugal machine: Biofuge28RS is available from German Heraeus (congratulating Li Shi) company;
Ultralow Temperature Freezer (80 ℃): available from Japanese Sanyo (Sanyo) company;
Constant incubator: available from Japanese Sanyo (Sanyo) company;
The pure water instrument: EASYPure UF07412, available from Millipore company (Mi Libo, the U.S.);
High-pressure sterilizing pot: LaboAut ℃ lave is available from Sanyo company (Sanyo, Japan);
Vertical electrophoresis system: MiniPROTEAN2﹠amp; 3Cell is available from U.S. Bio-Rad (Bole) company;
Inverted microscope: Lx50 Olympus, available from Japanese Olympus (Olympus) company;
Protein purification system:
Explorer is available from U.S. GE (general) company.
Embodiment one. anti-mouse of rabbit or the full myeloma cell's of people Polyclonal Antibody Preparation and evaluation
1, anti-mouse of rabbit or the full myeloma cell's of people Polyclonal Antibody Preparation
Full cell of myelomatosis (mouse MPC-11 or people ARH-77) that utilization is lived and complete Freund's adjuvant immunity new zealand white rabbit, get blood 1ml from the auricular vein of new zealand white rabbit after 7～14 days, solidify back centrifuging and taking serum, the ELISA method detects the titre of polyclonal antibody, it is 1: 2000, the back full freund's adjuvant that tood many or too much for use every 7～14 days mixes booster immunization with the full myeloma cell who lives, and all gets blood 1ml from auricular vein before each immunity, and the 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 contain in glass triangle flask (250ml), place 2～3h under the room temperature, then 4 degrees centigrade of refrigerator overnight.Next day, draw the supernatant in the Erlenmeyer flask, 2000 centrifugal 10 minutes, get supernatant, at inferior 10000rpm centrifugal 10 minutes, get supernatant and carry out antibody purification, perhaps-80 degree centigrade frozen to antibody purification.
The purifying of IgG antibody carries out according to the Mabselect specification sheets.In brief, pillar is chosen XK16/40, and filler is selected Mabselect for use.The purification system utilization
The explorer system.In conjunction with Buffer A (20mM NaH
2PO
4, 0.15M NaCl, pH 7.2) packing of centrifugal collection antibody serum, adopting immunoglobulin IgG in the Mabselect purified rabbit serum, the Bio-Rad method is measured antibody concentration and is reached 20mg/ml, Freeze Drying Equipment freeze-drying after the packing, and preserve-80 ℃.
Concrete purification process is:
Buffer A (20mM NaH with 10 times of pillar volumes
2PO
4, 0.15M NaCl, pH 7.2pH 9.0) and balance XK16/40 pillar;
Last sample immunize rabbit serum or contrast blank rabbit anteserum (50ml);
BufferA with 5 times of column volumes washes pillar again;
Wash post with linear gradient Buffer B (the 0.1M citric acid is received, and pH 3.0), until the appearance that elution peak is arranged;
Collect elution peak liquid, in using immediately then and Buffer (1M Tris-HCl is pH8.0) about adjusting eluting liquid pH7.2.
Use dialysis tubing as for PBS (PH7.2) dialysed overnight in 4 degrees centigrade of chromatography cabinets the elutriant of collecting then.
The collection liquid of dialysed overnight is obtained the IgG powder of purifying with the refrigerator freeze-drying, and-80 degrees centigrade of refrigerators are preserved.
Above method is suitable for various mouse or human myeloma cell extracts Polyclonal Antibody Preparation as antigen-immunized animal especially rabbit affinity chromatography.
2, the evaluation of anti-mouse of rabbit or the full myeloma cell's of people polyclonal antibody
1) cell ELISA: the myeloma cell is layered in 96 orifice plates of poly-lysine bag quilt by 10000 amount, and with not immune rabbit igg and polyclonal antibody (immune rabbit igg) by 1: 2000,1: 5000,1: 10000, make an antibody gradient, be ELISA at 1: 20000; (Fig. 1)
The results are shown in Figure 1, the result shows that antibody group of the present invention is 8～10 times of the control antibodies binding ability in conjunction with myeloma cell's ability, and has dose-dependent effect, along with the increase antibody group of Dilution ratio decreases in conjunction with the myelomatosis ability.
2) immunoblotting: collect 10
6Cell pyrolysis liquid is pressed sample electrophoresis on the different concns, change film, 5% skim-milk is as confining liquid, room temperature closed protein electricity changeed film 1 hour, the full myeloma cell's polyclonal antibody of the anti-people of rabbit concentration is that 5 μ g/ μ l are anti-as one, dilution in 1: 1000～1: 5000, 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 and punching exposure.
The results are shown in Figure 2, the result shows that polyclonal antibody can effectively discern different multiple myeloma cells system, however from protein band show polyclonal antibody the different myeloma cell line ability of identification difference (from left to right: MPC-11, SP2/0, NS-1).
3) cellular immunofluorescence dyeing: with 10
6Cell is fixed with 100 μ l4% Paraformaldehyde 96s, smear is on the slide glass that poly-lysine was handled then, 10% lowlenthal serum room temperature closing cell 1 hour, the full myeloma cell's polyclonal antibody of the anti-people of rabbit concentration 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 preceding 5 minutes, and hoechest dyes nuclear, and 1 * PBS shakes and washes 3 times, each 5～15 minutes; The mounting mirror is observed down; (Fig. 3)
The results are shown in Figure 3, the result shows, uses antibody group of the present invention, can see green fluorescence at the MPC-11 cell surface, and contrast normal rabbit serum immunoglobulin (Ig) then 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 discerned.
4) cell streaming: with the myeloma cell 10 of logarithmic phase
6With full myeloma cell's polyclonal antibody 5 μ g/ μ l incubated at room of the anti-people 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 with 1 * PBS washes 3 times, and each 5～15 minutes, at least 10000 cell upflowing cell instruments detections.(Fig. 4)
The results are shown in Figure 4, on behalf of mouse of the present invention source or human antibody group, gray line react with corresponding mouse source or people source multiple myeloma cells respectively, on behalf of control antibodies, black line react as negative control with mouse source or people source multiple myeloma cells respectively, the result shows, the all oriented right avertence of antibody group fluorescence intensity of the present invention is moved, and proves that antibody group of the present invention can effectively discern multiple multiple myeloma cells.(on arrange mouse myeloma cell line, from left to right: MPC-11, SP2/0, NS-1, following 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 two anti-myeloma cells
At first with 21 Babl/c mouse peritoneal inoculations 10
6About the MPC-11 cell, after 5 days it is divided into three groups, blank group (PBS); Control antibodies group (non-immune rabbit igg); Experimental group (rabbit igg of immunity is the polyclonal antibody group), 7 every group.Distinguish abdominal injection PBS then, not immune rabbit igg (200 μ g/ml) or polyclonal antibody (200 μ g/ml), injection every other day once has altogether six times.After the 6th injection, put to death mouse in second day, 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 bigger, magnitude range is about 0.2～1.5cm, and antibody group mouse peritoneal tubercle magnitude range of the present invention is about 0.1～0.6cm, and the result shows that antibody of the present invention has the effect that obvious inhibition multiple myeloma is grown in the mouse body.
Embodiment three. tumor killing effect in the body of the polyclonal antibody of xenotransplantation anti-myeloma cell
At first with the right back of the body of 21 SCID mouse subcutaneous abdomen inoculation 10
6About people source multiple myeloma cells ARH-77, when laying one's hand on and it being divided into three groups, blank group (NS) at random during tumor nodule; Control antibodies group (non-immune rabbit igg); Experimental group (rabbit igg of immunity is the polyclonal antibody group), 7 every group.Distinguish tail vein injection NS then, not immune rabbit igg (200 μ g/ml) or polyclonal antibody (200 μ g/ml) 100 microlitres, injection every other day once has altogether six times.To suppress effect in order verifying in the body of antibody group of the present invention to the xenotransplantation tumour, to use the vernier caliper measurement gross tumor volume, every other day measure once, draw tumor growth curve.
The results are shown in Figure 6, ◆, ■, ▲ represent NS respectively, control antibodies and of the present invention group of antibody treatment mouse tumor growth volume, the result shows that antibody group of the present invention is handled mouse can obviously suppress growth in the tumour body.
The polyclonal antibody safety evaluation of embodiment four anti-myeloma cells of the present invention
Get and accept polyclonal antibody treatment (200 μ g/ml) group, with the heart of the mouse that gives control antibodies group and physiological saline group, liver, spleen, lung and nephridial tissue, carry out HE dyeing, whether observation gives polyclonal antibody treatment back main organs damage.Further by using the MTT method
The results are shown in Figure 7, the result shows antibody group mouse core of the present invention, liver, and spleen, lung is compared with the physiological saline group with the control antibodies group with nephridial tissue, does not have tangible pathological characters and changes.
Get the normal mouse splenocyte, the control antibodies and the polyclonal antibody (200g/ml) that give physiological saline, same concentrations are respectively handled, and analyze by MTT after 48 hours, observe the cytotoxicity of polyclonal antibody processing to normal mouse boosting cell.
The results are shown in Figure 8, antibody of the present invention does not show any cytotoxic effect to splenocyte effect and the physiological saline and the control antibodies treatment effect basically identical of normal mouse.
Claims (7)
1. myeloma cell's polyclonal antibody is characterized in that: with making behind the myeloma cell immune animal.
2. myeloma cell's polyclonal antibody according to claim 1 is characterized in that it being to be made by following method:
A, use 1 * 10 first
6～5 * 10
7The myeloma cell of individual work and complete Freund's adjuvant immune animal;
B, then once, booster immunization too many or too much for use full freund's adjuvant and myeloma cell's mixed immunity of living every 7～14 days booster immunizations; All detect the blood antibody titers before each immunity, when antibody titers reaches 1: 10000～termination immunity more than 50000;
C, stop getting after the immunity blood and spend the night in 4 degrees centigrade after placing 2～3h under the room temperature, get the centrifugal serum that obtains containing myeloma cell's polyclonal antibody of supernatant then, purifying makes myeloma cell's polyclonal antibody from the serum that contains myeloma cell's polyclonal antibody.
Claim 1 or 2 described myeloma cell's polyclonal antibodies the preparation diagnose multiple myeloma reagent in purposes.
4. claim 1 or the 2 described myeloma cell's polyclonal antibodies purposes in the medicine of preparation treatment multiple myeloma.
5. the method for purifying myeloma cell polyclonal antibody is characterized in that:
A, the serum that will contain myeloma cell's polyclonal antibody are splined on the buffer A equilibrated pillar with 10 times of column volumes, and the pillar filler is Mabselect, washes pillar with the buffer A of 5 times of column volumes again; Described buffer A is 20mM NaH
2PO
4, 0.15M NaCl, pH 7.2;
B, make the buffer B linear gradient with the 0.1M Trisodium Citrate of pH 3.0 and wash pillar, until the appearance that elution peak is arranged;
C, collect elution peak liquid, the Tris-HCl with pH8.0,1M does neutralization buffer simultaneously, and the adjusting elutriant is to the pH7.2;
D, the elutriant of collecting is placed PH7.2 with dialysis tubing then the PBS damping fluid in 4 dialysis down Celsius, the collection liquid freeze-drying of dialysis is promptly got myeloma cell's polyclonal antibody.
6. the method for preparing myeloma cell's polyclonal antibody is characterized in that may further comprise the steps:
A, use 1 * 10 first
6～5 * 10
7The myeloma cell of individual work and complete Freund's adjuvant immune animal;
B, then once, booster immunization too many or too much for use full freund's adjuvant and myeloma cell's mixed immunity of living every 7～14 days booster immunizations; All detect the blood antibody titers before each immunity, when antibody titers reaches 1: 10000～termination immunity more than 50000;
C, stop getting after the immunity blood and after placing 2～3h under the room temperature, spend the night in 4 degrees centigrade, get the centrifugal serum that obtains containing myeloma cell's polyclonal antibody of supernatant then, make myeloma cell's polyclonal antibody from the serum purifying.
7. the method for preparing myeloma cell's polyclonal antibody according to claim 6 is characterized in that the purifying in the described steps d may further comprise the steps:
The serum that will contain myeloma cell's polyclonal antibody is splined on the buffer A equilibrated XK16/40 post with 10 times of column volumes, and filler is Mabselect;
Wash pillar with the buffer A of 5 times of column volumes again; Wash post with the buffer B linear gradient, until the appearance that elution peak is arranged; Collect elution peak liquid, regulate about elutriant pH7.2 with neutralization buffer simultaneously;
Place PBS damping fluid (PH7.2) 4 degrees centigrade of dialysis down with dialysis tubing the elutriant of collecting, the collection liquid freeze-drying of dialysing is promptly got myeloma cell's polyclonal antibody;
Above-mentioned buffer A is 20mM NaH
2PO
4, 0.15M NaCl, pH 7.2pH 9.0; Buffer B is the 0.1M Trisodium Citrate, and pH 3.0; Neutralization buffer is 1M Tris-HCl, pH8.0.
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