CN117720641A - Preparation method of drug-loaded human serum albumin combined monoclonal antibody - Google Patents
Preparation method of drug-loaded human serum albumin combined monoclonal antibody Download PDFInfo
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Abstract
The invention discloses a preparation method of a drug-loaded human serum albumin combined monoclonal antibody; s1: antigen, namely, antigen (1) hybridoma can survive and passage in vitro in a 'permanent' way, can generate high-specificity and high-uniformity antibody (2) continuously by using relatively impure antigen as long as no gene mutation of cell lines occurs, and can obtain a large amount of high-specificity and uniform antibodies; (3) Since it is possible to obtain "unlimited amounts" of homogeneous antibodies, it is suitable for immunological analytical methods featuring labeled antibodies, such as IRMA and ELISA, etc.; (4) Due to the high specificity and single biological function of monoclonal antibodies, the monoclonal antibodies can be used for in vivo radioimmunoimaging and immunoguided therapy.
Description
Technical Field
The invention relates to the technical field of monoclonal antibody preparation, in particular to a preparation method of a drug-loaded human serum albumin combined monoclonal antibody.
Background
Molecular biologists g.j.f. kler and c.millstein in 1975 created a vertical hybridoma technique based on natural hybridization techniques. They fused myeloma cells, which can be cultured in vitro and immortalized, with the spleen cells of naive mice after immunization with antigen to form B cell hybridomas. The hybridoma cell has the characteristic that the tumor cell is easy to proliferate in vitro and has the characteristic that the spleen lymphocyte can synthesize and secrete specific antibody.
Such hybridomas, by cloning, can give rise to monoclonal lines from individual hybridoma cells, i.e. hybridoma cell lines, which produce antibodies which are highly homogeneous against the same epitope, so-called monoclonal antibodies, abbreviated as monoclonal antibodies. Monoclonal antibodies are produced by lymphocytic hybridomas and are directed against only a single epitope on a complex antigen molecule. Monoclonal antibody technology (monoclon aliibodytechnique): an immunological technique for hybridizing single B lymphocyte to generate antibody with bone marrow tumor cells to obtain the hybrid cell able to generate antibody and unlimited proliferation, and generating antibody. Is an antibody produced by only one type of cell, and corresponds to a polyclonal antibody or a polyclonal antibody, which is an antibody produced by a plurality of types of cells; (1) The inherent affinity and limited biological activity of monoclonal antibodies limit its range of applications. Since monoclonal antibodies are not amenable to precipitation and agglutination reactions, many detection methods cannot be accomplished with monoclonal antibodies. (2) Monoclonal antibodies are not as reactive as polyclonal antibodies. (3) The preparation technique is complex, time-consuming and labor-consuming, so that the monoclonal antibody is also relatively expensive.
Disclosure of Invention
The invention solves the technical problem of overcoming the prior art of providing a preparation method of a drug-loaded human serum albumin combined monoclonal antibody.
In order to achieve the above purpose, the present invention provides the following technical solutions: the preparation method of the drug-loaded human serum albumin combined monoclonal antibody comprises the following steps:
s1: an antigen refers to a substance which can stimulate an organism to generate (specific) immune response, and can combine with immune response product antibodies and sensitized lymphocytes in vitro to generate immune effects (specific reaction); the basic properties of an antigen are two, namely, the ability to induce an immune response, i.e., immunogenicity, and the ability to react with the products of an immune response, i.e., antigenicity; many substances can be used as antigens, the specific classification of the antigens can be seen as antigens, in the process of preparing monoclonal antibodies, many substances can be used as antigens, in the conventional scientific experiments, researchers often select 10-50 ug of recombinant protein, coupled polypeptide, coupled small molecule and the like to be injected into each mouse/rat as antigens to generate specific monoclonal antibodies;
s2: selection and immunization of animals
(1) The pure BALB/c mice are selected from animals, which are warm and smooth, have small range of motion from nest, weak body, small food intake and pollution discharge, and the laboratory with clean environment can be used for raising the pure BALA/c mice in the laboratory for developing hybridoma technology;
(2) the selection of an appropriate immunization scheme is important for the success of cell fusion hybridization and the acquisition of high-quality McAb; generally, the primary immunization is started about two months before fusion according to established immunization schemes, and the immunization schemes are determined according to different characteristics of antigens;
(1) The immunogenicity of the soluble antigen is weaker, and usually, an adjuvant is added, and hapten should be firstly prepared into immunogen and then added with adjuvant;
(2) The particle antigen has strong immunity, and can obtain good immunity effect without adding adjuvant;
s3: preparation before cell fusion
(1) Selection of myeloma cell lines: myeloma cells and immune animals belong to the same strain, the sample hybridization fusion rate is high, and the hybridoma can be inoculated conveniently to generate a McAb in the abdominal cavity of mice of the same strain;
(2) Feeder cells: in tissue culture, individual or few discrete cells are not prone to growth and proliferation, and if other living cells are added, the growth and proliferation of the cells can be promoted, and the added number of the cells is called feeder cells; in the preparation of mcabs, many procedures require the addition of feeder cells, such as during hybridoma cell selection, cloning and expansion culture, which are necessary; common feeder cells are: mouse peritoneal macrophages (more commonly used), mouse spleen cells or thymus cells; the amount of feeder cells is typically 2 x 104 or 105 cells/well;
s4: step of cell fusion
(1) Myeloma cells and spleen cells were mixed at a ratio of 1:10 or 1:5, washing with serum-free incomplete culture solution 1 time in a 50ml centrifuge tube, centrifuging at 1200rpm for 8min; discarding the supernatant, and sucking the residual liquid with a suction tube to avoid affecting polyethylene glycol (PEG) concentration; lightly flicking the bottom of the centrifugal tube to slightly loosen the cell sediment;
(2) 1ml of 45% PEG (molecular weight 4000) solution pre-warmed at 37℃was added over 90s with gentle shaking; water bath at 37 ℃ for 90s;
(3) adding 37 ℃ pre-warmed incomplete culture solution to stop PEG effect, and adding 1ml, 2ml, 3ml, 4ml, 5ml and 6ml respectively every 2 min;
(4) centrifuging at 800rpm for 6min;
(5) filling the supernatant, and re-suspending the supernatant by using HAT selective culture solution containing 20% calf serum;
(6) the cells were added to a 96-well plate of an existing feeder cell layer, and 100. Mu.l of the cells were added to each well; typically, one immune spleen can be inoculated with 4 96-well plates;
(7) placing the culture plate in a culture box with 5% CO2 at 37 ℃ for culture;
s5: selection of hybridoma cells and antibody detection
S6: HAT selection of hybridoma cells
After the spleen cells and the myeloma cells are treated by PEG, a mixture of various cells is formed, and only hybridoma cells formed by the spleen cells and the myeloma cells are of significance;
s7: the method for detecting the antibody is to select different screening methods according to the nature of the antigen and the type of the antibody, and generally adopts a rapid, simple, specific and sensitive method as a principle;
s8: cryopreservation and resuscitation of hybridoma cells
S9: cryopreservation of hybridoma cells
It is important to freeze the subcloned cells obtained by cloning the hybridoma cells in the original hole each time in time; because contamination of cells, loss of antibody secretion ability, etc. may occur at any time during the culture of cells without establishing a stable antibody-secreting cell line; if the original cells are not frozen, the original cells can be discarded before the original cells are frozen; cell cryopreservation solution: 50% calf serum; 40% of incomplete culture solution; 10% dmso (dimethyl sulfoxide);
preferably, adjuvants commonly used in S2 (1): complete Freund's adjuvant, incomplete Freund's adjuvant; 1-50 mug of primary immunization antigen is injected subcutaneously or intraparenchymally (generally 0.8-1 ml,0.2 ml/spot) with Freund's complete adjuvant, 3 weeks later, second immunization is carried out, the dosage is the same as above, the Freund's incomplete adjuvant is added subcutaneously or ip (intraperitoneal injection) (ip dosage is not more than 0.5 ml), 3 weeks later, third immunization is carried out, the dosage is the same, no adjuvant is added, ip (blood is taken after 5-7 days for measuring the titer), 2-3 weeks is carried out for boosting immunization, 50-500 mug of dosage is suitable, ip or iv (intravenous injection) is carried out, and spleen fusion is carried out after 3 days.
Preferably, S2 (2) is exemplified by a cellular antigen, and the amount of antigen required for immunization is 1 to 2X 107 cells; after 1X 107/0.5mlip for 2-3 weeks of primary immunization, 1X 107/0.5mlip for the secondary immunization and 1X 107/0.5mlip or iv for 3 weeks of post boost (three days before fusion), spleen was taken for fusion.
Preferably, the myeloma cells in S3 can be cultured in a common culture medium such as DMEM medium; the concentration of calf serum is generally 10% -20%, the cell concentration is preferably 104-5×105/ml, and the maximum concentration is not more than 106/ml; when the cell is in the middle stage of growth, the cell can be grown as follows: 3-1: 10, passaging in proportion; passaging every 3-5 days; during the passage of cells, partial cells may have the phenomenon of reversion, and 8-azaguanine should be periodically used for treatment, so that living cells have uniform sensitivity to HAT.
Preferably, when cultured in HAT selective medium in S6, myeloma cells lack thymidine kinase or hypoxanthine guanine ribose transferase and are therefore incapable of growing, whereas hybridoma cells have both enzymes and are capable of growing in HAT selective medium; culturing for 1-2 days by using HAT, wherein a large number of tumor cells die, the tumor cells disappear after 3-4 days, the hybridization cells form small colonies, and after maintaining for 7-10 days, HT culture solution is replaced by HAT selection culture solution, and then the HT culture solution is maintained for 2 weeks, and then general culture solution is used; during the selective culture period, when the hybridoma cells are distributed to be 1/10 of the area of the hole bottom, specific antibodies can be detected, and the needed hybridoma cell line is screened out; during the selection culture, half of the culture medium is generally changed every 2 to 3 days.
Preferably, the frozen stock solution in the step S9 is precooled, and the operation is gentle and rapid; can be immediately reduced to 0 from room temperature during freezing placing in a refrigerator with ultralow temperature of-70 ℃, transferring into liquid nitrogen the next day; freezing can also be carried out by using a cell freezing device; the frozen cells are periodically resuscitated, and the activity of the cells and the stability of the secreted antibodies are checked, and the cells can be preserved for several years or more in liquid nitrogen; 2.6.2 method of cell resuscitation glass ampoule is carefully removed from liquid nitrogen, put in 37 ℃ water bath, thawing frozen cells in 1min, washing the cells twice with complete culture solution, then transferring into culture flask of feeder cells prepared on the first day, culturing in 37 ℃ 5% CO2 incubator, and detecting antibody activity when the cells form colonies.
Compared with the prior art, the invention has the beneficial effects that:
(1) The hybridoma can survive and be passaged in vitro in a 'permanent' manner, and can continuously produce high-specificity and high-uniformity antibodies as long as the gene mutation of the cell strain does not occur
(2) A large number of highly specific, homogeneous antibodies can be obtained with relatively impure antigens.
(3) Since it is possible to obtain "unlimited amounts" of homogeneous antibodies, it is suitable for immunological analytical methods featuring labeled antibodies, such as IRMA and ELISA, etc. (4) Due to the high specificity and single biological function of monoclonal antibodies, the monoclonal antibodies can be used for in vivo radioimmunoimaging and immunoguided therapy.
In summary, the following are: high specificity, high purity, good repeatability, strong sensitivity, low cost, mass production and the like; 1.2 limitations of monoclonal antibodies:
first, hybrid myeloma cells are obtained; the hybrid cell inherits the characteristics of two parent cells, has the characteristic that B lymphocytes synthesize specific antibodies, and has the characteristic that myeloma cells can be cultured and proliferated in vitro for perpetuation, and the cell population derived from single fusion cells and cultured and proliferated can be used for preparing specific monoclonal antibodies against an antigenic determinant.
Drawings
FIG. 1 is a schematic diagram of the preparation of a monoclonal antibody of the invention;
FIG. 2 is a diagram of a conventional immunocyte program according to the present invention;
FIG. 3 is a diagram of cell fusion according to the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-3, the present invention provides a technical solution: the preparation method of the drug-loaded human serum albumin combined monoclonal antibody comprises the following steps:
s1: an antigen refers to a substance which can stimulate an organism to generate (specific) immune response, and can combine with immune response product antibodies and sensitized lymphocytes in vitro to generate immune effects (specific reaction); the basic properties of an antigen are two, namely, the ability to induce an immune response, i.e., immunogenicity, and the ability to react with the products of an immune response, i.e., antigenicity; many substances can be used as antigens, the specific classification of the antigens can be seen as antigens, in the process of preparing monoclonal antibodies, many substances can be used as antigens, in the conventional scientific experiments, researchers often select 10-50 ug of recombinant protein, coupled polypeptide, coupled small molecule and the like to be injected into each mouse/rat as antigens to generate specific monoclonal antibodies;
s2: selection and immunization of animals
(1) The pure BALB/c mice are selected from animals, which are warm and smooth, have small range of motion from nest, weak body, small food intake and pollution discharge, and the laboratory with clean environment can be used for raising the pure BALA/c mice in the laboratory for developing hybridoma technology;
(2) the selection of an appropriate immunization scheme is important for the success of cell fusion hybridization and the acquisition of high-quality McAb; generally, the primary immunization is started about two months before fusion according to established immunization schemes, and the immunization schemes are determined according to different characteristics of antigens;
(1) The immunogenicity of the soluble antigen is weaker, and usually, an adjuvant is added, and hapten should be firstly prepared into immunogen and then added with adjuvant;
(2) The particle antigen has strong immunity, and can obtain good immunity effect without adding adjuvant;
s3: preparation before cell fusion
(1) Selection of myeloma cell lines: myeloma cells and immune animals belong to the same strain, the sample hybridization fusion rate is high, and the hybridoma can be inoculated conveniently to generate a McAb in the abdominal cavity of mice of the same strain;
(2) Feeder cells: in tissue culture, individual or few discrete cells are not prone to growth and proliferation, and if other living cells are added, the growth and proliferation of the cells can be promoted, and the added number of the cells is called feeder cells; in the preparation of mcabs, many procedures require the addition of feeder cells, such as during hybridoma cell selection, cloning and expansion culture, which are necessary; common feeder cells are: mouse peritoneal macrophages (more commonly used), mouse spleen cells or thymus cells; the amount of feeder cells is typically 2 x 104 or 105 cells/well;
s4: step of cell fusion
(1) Myeloma cells and spleen cells were mixed at a ratio of 1:10 or 1:5, washing with serum-free incomplete culture solution 1 time in a 50ml centrifuge tube, centrifuging at 1200rpm for 8min; discarding the supernatant, and sucking the residual liquid with a suction tube to avoid affecting polyethylene glycol (PEG) concentration; lightly flicking the bottom of the centrifugal tube to slightly loosen the cell sediment;
(2) 1ml of 45% PEG (molecular weight 4000) solution pre-warmed at 37℃was added over 90s with gentle shaking; water bath at 37 ℃ for 90s;
(3) adding 37 ℃ pre-warmed incomplete culture solution to stop PEG effect, and adding 1ml, 2ml, 3ml, 4ml, 5ml and 6ml respectively every 2 min;
(4) centrifuging at 800rpm for 6min;
(5) filling the supernatant, and re-suspending the supernatant by using HAT selective culture solution containing 20% calf serum;
(6) the cells were added to a 96-well plate of an existing feeder cell layer, and 100. Mu.l of the cells were added to each well; typically, one immune spleen can be inoculated with 4 96-well plates;
(7) placing the culture plate in a culture box with 5% CO2 at 37 ℃ for culture;
s5: selection of hybridoma cells and antibody detection
S6: HAT selection of hybridoma cells
After the spleen cells and the myeloma cells are treated by PEG, a mixture of various cells is formed, and only hybridoma cells formed by the spleen cells and the myeloma cells are of significance;
s7: the method for detecting the antibody is to select different screening methods according to the nature of the antigen and the type of the antibody, and generally adopts a rapid, simple, specific and sensitive method as a principle;
s8: cryopreservation and resuscitation of hybridoma cells
S9: cryopreservation of hybridoma cells
It is important to freeze the subcloned cells obtained by cloning the hybridoma cells in the original hole each time in time; because contamination of cells, loss of antibody secretion ability, etc. may occur at any time during the culture of cells without establishing a stable antibody-secreting cell line; if the original cells are not frozen, the original cells can be discarded before the original cells are frozen; cell cryopreservation solution: 50% calf serum; 40% of incomplete culture solution; 10% dmso (dimethyl sulfoxide);
preferably, adjuvants commonly used in S2 (1): complete Freund's adjuvant, incomplete Freund's adjuvant; 1-50 mug of primary immunization antigen is injected subcutaneously or intraparenchymally (generally 0.8-1 ml,0.2 ml/spot) with Freund's complete adjuvant, 3 weeks later, second immunization is carried out, the dosage is the same as above, the Freund's incomplete adjuvant is added subcutaneously or ip (intraperitoneal injection) (ip dosage is not more than 0.5 ml), 3 weeks later, third immunization is carried out, the dosage is the same, no adjuvant is added, ip (blood is taken after 5-7 days for measuring the titer), 2-3 weeks is carried out for boosting immunization, 50-500 mug of dosage is suitable, ip or iv (intravenous injection) is carried out, and spleen fusion is carried out after 3 days.
Preferably, S2 (2) is exemplified by a cellular antigen, and the amount of antigen required for immunization is 1 to 2X 107 cells; after 1X 107/0.5mlip for 2-3 weeks of primary immunization, 1X 107/0.5mlip for the secondary immunization and 1X 107/0.5mlip or iv for 3 weeks of post boost (three days before fusion), spleen was taken for fusion.
Preferably, the myeloma cells in S3 can be cultured in a common culture medium such as DMEM medium; the concentration of calf serum is generally 10% -20%, the cell concentration is preferably 104-5×105/ml, and the maximum concentration is not more than 106/ml; when the cell is in the middle stage of growth, the cell can be grown as follows: 3-1: 10, passaging in proportion; passaging every 3-5 days; during the passage of cells, partial cells may have the phenomenon of reversion, and 8-azaguanine should be periodically used for treatment, so that living cells have uniform sensitivity to HAT.
Preferably, when cultured in HAT selective medium in S6, myeloma cells lack thymidine kinase or hypoxanthine guanine ribose transferase and are therefore incapable of growing, whereas hybridoma cells have both enzymes and are capable of growing in HAT selective medium; culturing for 1-2 days by using HAT, wherein a large number of tumor cells die, the tumor cells disappear after 3-4 days, the hybridization cells form small colonies, and after maintaining for 7-10 days, HT culture solution is replaced by HAT selection culture solution, and then the HT culture solution is maintained for 2 weeks, and then general culture solution is used; during the selective culture period, when the hybridoma cells are distributed to be 1/10 of the area of the hole bottom, specific antibodies can be detected, and the needed hybridoma cell line is screened out; during the selection culture, half of the culture medium is generally changed every 2 to 3 days.
Preferably, the frozen stock solution in the step S9 is precooled, and the operation is gentle and rapid; can be immediately reduced to 0 from room temperature during freezing placing in a refrigerator with ultralow temperature of-70 ℃, transferring into liquid nitrogen the next day; freezing can also be carried out by using a cell freezing device; the frozen cells are periodically resuscitated, and the activity of the cells and the stability of the secreted antibodies are checked, and the cells can be preserved for several years or more in liquid nitrogen; 2.6.2 method of cell resuscitation glass ampoule is carefully removed from liquid nitrogen, put in 37 ℃ water bath, thawing frozen cells in 1min, washing the cells twice with complete culture solution, then transferring into culture flask of feeder cells prepared on the first day, culturing in 37 ℃ 5% CO2 incubator, and detecting antibody activity when the cells form colonies.
Embodiment two: the preparation method of drug-loaded human serum albumin combined monoclonal antibody comprises the following steps of:
there are two main methods for mass production of monoclonal antibodies: one is in vitro culture of hybridoma cells, and monoclonal antibodies are isolated in the culture solution; the method needs special instruments and equipment, and generally uses serum-free culture medium to facilitate concentration and purification of monoclonal antibodies; if mass produced, the cost is high;
another method is most commonly employed in the intraperitoneal vaccination of mice; selecting BALB/c mice or parent mice thereof, injecting the mice into the abdominal cavity by using pristane or liquid paraffin, and inoculating hybridoma cells into the abdominal cavity after one week; usually, after one week of inoculation, obvious ascites can be generated, and 5-10 ml of ascites can be collected for each mouse, and sometimes even more than 40ml of ascites can be collected; the ascites antibody prepared by the method has high content, and can reach the level of milligrams or even tens of milligrams per milliliter; in addition, the ascites has less impurity protein, which is convenient for the purification of the antibody;
identification of monoclonal antibodies is necessary for systematic identification of the prepared McAb, and should be accomplished in several ways: identification of antibody specificity in addition to detection of antibodies with an immunogen (antigen), cross-test should be performed with other antigens related to their antigen components by ELISA, IFA;
for example: (1) preparing a McAb for resisting melanoma cells, and performing cross reaction by using tumor cells and normal cells of other organs besides melanoma cells so as to select monoclonal antibodies of tumor specific or tumor related antigens; (2) preparing a monoclonal antibody of the recombinant cytokine, firstly considering whether the monoclonal antibody has cross reaction with the protein of the expression strain, and secondly considering whether the monoclonal antibody has cross reaction with other cytokines;
identification of the Ig class and subclass of mcabs generally the Ig class of antibodies has been substantially determined upon screening with enzyme-labeled or fluorescein-labeled secondary antibodies; if enzyme-labeled or fluorescein-labeled rabbit anti-mouse IgG or IgM is used, the antibodies detected are typically of the IgG or IgM class; as subclasses, it is necessary to use standard anti-subclass serum systems for double-amplification or sandwich ELISA; in the double-amplification test, if a proper amount of PEG (3%) is added, the formation of a precipitation line is more beneficial
Identification of neutralizing Activity biological activity of McAb was determined by animal or cell protection experiments; for example, if the neutralizing activity of an antiviral McAb is determined, then the antibody and virus can be simultaneously inoculated into a susceptible animal or susceptible cell to see if the animal or cell is protected by the antibody;
identification of McAb recognized epitope Using competitive binding assay, method of measuring addition index, determining the antigen site recognized by McAb to determine whether the recognized epitope of McAb is identical;
identification of McAb avidity was determined for binding to the corresponding antigen using ELISA or RIA competition binding assays;
influence factors of monoclonal antibody preparation:
1. pollution;
2. PEG is toxic, the possible action time is too long, the bovine serum quality is poor, myeloma cells pollute mycoplasma, HAT has problems, and the hybridoma cells do not grow after fusion; 3, the immunogenicity is weak, the immune effect is poor, cells grow after fusion, but no antibody is produced (the A in HAT is invalid or myeloma cells are mutated and become A resisting cells), and the hybridoma cells which originally secrete the antibody become negative (the mycoplasma pollution or the non-antibody secreting cells are cloned and competitively grown), so that the hybridoma cells do not secrete the antibody or stop secreting the antibody; 4 calf serum quality problem, hybridoma cell activity state, mycoplasma pollution, hard cloning of hybridoma cell, etc.
Embodiment III: the preparation method of the drug-loaded human serum albumin combined monoclonal antibody comprises the following steps: use of monoclonal antibodies:
1. currently, commercial kits made using monoclonal antibodies are widely used for testing medical diagnostic reagents:
(1) detecting pathogenic microorganism antigens and antibodies; (2) detecting tumor antigens; (3) detection of immune cells and subpopulations thereof; (4) hormone determination; (5) measurement of cytokines; the antigen recognition by monoclonal antibodies is very different from that by polyclonal antibodies; different kits have different detection results due to different monoclonal antibodies and different antigen recognition sites; therefore, the standardization problem still requires further investigation;
2. purification of proteins monoclonal antibodies are important ligands in affinity chromatography; adsorbing the monoclonal antibody on an inert solid-phase matrix and preparing a chromatographic column; when the sample flows through the chromatographic column, the antigen to be separated can be specifically combined with the monoclonal antibody of the solid phase, and the rest components can not be combined with the monoclonal antibody; after the chromatographic column is fully eluted, changing the ionic strength or pH of the eluent, dissociating the antigen to be separated from the antibody, and collecting the eluent to obtain the antigen to be purified;
3. the tumor guiding treatment and radioimmunoimaging technology connects a monoclonal antibody aiming at a certain tumor antigen with a chemotherapeutic drug or a radiotherapy substance, and the drug or the radiotherapy substance is carried to a target organ by utilizing the guiding action of the monoclonal antibody to directly kill target cells, which is called tumor guiding treatment.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. The preparation method of the drug-loaded human serum albumin combined monoclonal antibody comprises the steps of: the method comprises the following steps:
s1: an antigen refers to a substance which can stimulate an organism to generate (specific) immune response, and can combine with immune response product antibodies and sensitized lymphocytes in vitro to generate immune effects (specific reaction); the basic properties of an antigen are two, namely, the ability to induce an immune response, i.e., immunogenicity, and the ability to react with the products of an immune response, i.e., antigenicity; many substances can be used as antigens, the specific classification of the antigens can be seen as antigens, in the process of preparing monoclonal antibodies, many substances can be used as antigens, in the conventional scientific experiments, researchers often select 10-50 ug of recombinant protein, coupled polypeptide, coupled small molecule and the like to be injected into each mouse/rat as antigens to generate specific monoclonal antibodies;
s2: selection and immunization of animals
(1) The pure BALB/c mice are selected from animals, which are warm and smooth, have small range of motion from nest, weak body, small food intake and pollution discharge, and the laboratory with clean environment can be used for raising the pure BALA/c mice in the laboratory for developing hybridoma technology;
(2) the selection of an appropriate immunization scheme is important for the success of cell fusion hybridization and the acquisition of high-quality McAb; generally, the primary immunization is started about two months before fusion according to established immunization schemes, and the immunization schemes are determined according to different characteristics of antigens;
(1) The immunogenicity of the soluble antigen is weaker, and usually, an adjuvant is added, and hapten should be firstly prepared into immunogen and then added with adjuvant;
(2) The particle antigen has strong immunity, and can obtain good immunity effect without adding adjuvant;
s3: preparation before cell fusion
(1) Selection of myeloma cell lines: myeloma cells and immune animals belong to the same strain, the sample hybridization fusion rate is high, and the hybridoma can be inoculated conveniently to generate a McAb in the abdominal cavity of mice of the same strain;
(2) Feeder cells: in tissue culture, individual or few discrete cells are not prone to growth and proliferation, and if other living cells are added, the growth and proliferation of the cells can be promoted, and the added number of the cells is called feeder cells; in the preparation of mcabs, many procedures require the addition of feeder cells, such as during hybridoma cell selection, cloning and expansion culture, which are necessary; common feeder cells are: mouse peritoneal macrophages (more commonly used), mouse spleen cells or thymus cells; the amount of feeder cells is typically 2 x 104 or 105 cells/well;
s4: step of cell fusion
(1) Myeloma cells and spleen cells were mixed at a ratio of 1:10 or 1:5, washing with serum-free incomplete culture solution 1 time in a 50ml centrifuge tube, centrifuging at 1200rpm for 8min; discarding the supernatant, and sucking the residual liquid with a suction tube to avoid affecting polyethylene glycol (PEG) concentration; lightly flicking the bottom of the centrifugal tube to slightly loosen the cell sediment;
(2) 1ml of 45% PEG (molecular weight 4000) solution pre-warmed at 37℃was added over 90s with gentle shaking; water bath at 37 ℃ for 90s;
(3) adding 37 ℃ pre-warmed incomplete culture solution to stop PEG effect, and adding 1ml, 2ml, 3ml, 4ml, 5ml and 6ml respectively every 2 min;
(4) centrifuging at 800rpm for 6min;
(5) filling the supernatant, and re-suspending the supernatant by using HAT selective culture solution containing 20% calf serum;
(6) the cells were added to a 96-well plate of an existing feeder cell layer, and 100. Mu.l of the cells were added to each well; typically, one immune spleen can be inoculated with 4 96-well plates;
(7) placing the culture plate in a culture box with 5% CO2 at 37 ℃ for culture;
s5: selection of hybridoma cells and antibody detection
S6: HAT selection of hybridoma cells
After the spleen cells and the myeloma cells are treated by PEG, a mixture of various cells is formed, and only hybridoma cells formed by the spleen cells and the myeloma cells are of significance;
s7: the method for detecting the antibody is to select different screening methods according to the nature of the antigen and the type of the antibody, and generally adopts a rapid, simple, specific and sensitive method as a principle;
s8: cryopreservation and resuscitation of hybridoma cells
S9: cryopreservation of hybridoma cells
It is important to freeze the subcloned cells obtained by cloning the hybridoma cells in the original hole each time in time; because contamination of cells, loss of antibody secretion ability, etc. may occur at any time during the culture of cells without establishing a stable antibody-secreting cell line; if the original cells are not frozen, the original cells can be discarded before the original cells are frozen;
cell cryopreservation solution: 50% calf serum; 40% of incomplete culture solution; 10% DMSO (dimethyl sulfoxide).
2. The method for preparing the drug-loaded human serum albumin combined monoclonal antibody according to claim 1, wherein the method comprises the following steps of: adjuvants commonly used in S2 (1): complete Freund's adjuvant, incomplete Freund's adjuvant; 1-50 mug of primary immunization antigen is injected subcutaneously or intraparenchymally (generally 0.8-1 ml,0.2 ml/spot) with Freund's complete adjuvant, 3 weeks later, second immunization is carried out, the dosage is the same as above, the Freund's incomplete adjuvant is added subcutaneously or ip (intraperitoneal injection) (ip dosage is not more than 0.5 ml), 3 weeks later, third immunization is carried out, the dosage is the same, no adjuvant is added, ip (blood is taken after 5-7 days for measuring the titer), 2-3 weeks is carried out for boosting immunization, 50-500 mug of dosage is suitable, ip or iv (intravenous injection) is carried out, and spleen fusion is carried out after 3 days.
3. The method for preparing the drug-loaded human serum albumin combined monoclonal antibody according to claim 1, wherein the method comprises the following steps of: in S2, (2) the antigen is exemplified by a cellular antigen, and the amount of antigen required for immunization is 1 to 2X 107 cells; after 1X 107/0.5mlip for 2-3 weeks of primary immunization, 1X 107/0.5mlip for the secondary immunization and 1X 107/0.5mlip or iv for 3 weeks of post boost (three days before fusion), spleen was taken for fusion.
4. The method for preparing the drug-loaded human serum albumin combined monoclonal antibody according to claim 1, wherein the method comprises the following steps of: culturing myeloma cells in S3 using a common culture medium such as DMEM medium; the concentration of calf serum is generally 10% -20%, the cell concentration is preferably 104-5×105/ml, and the maximum concentration is not more than 106/ml; when the cell is in the middle stage of growth, the cell can be grown as follows: 3-1: 10, passaging in proportion; passaging every 3-5 days; during the passage of cells, partial cells may have the phenomenon of reversion, and 8-azaguanine should be periodically used for treatment, so that living cells have uniform sensitivity to HAT.
5. The method for preparing the drug-loaded human serum albumin combined monoclonal antibody according to claim 1, wherein the method comprises the following steps of: s6, when the myeloma cells are cultured in the HAT selection culture solution, the myeloma cells lack thymidine kinase or hypoxanthine guanine ribose transferase and cannot grow and reproduce, and the hybridoma cells have the two enzymes and can grow and reproduce in the HAT selection culture solution; culturing for 1-2 days by using HAT, wherein a large number of tumor cells die, the tumor cells disappear after 3-4 days, the hybridization cells form small colonies, and after maintaining for 7-10 days, HT culture solution is replaced by HAT selection culture solution, and then the HT culture solution is maintained for 2 weeks, and then general culture solution is used; during the selective culture period, when the hybridoma cells are distributed to be 1/10 of the area of the hole bottom, specific antibodies can be detected, and the needed hybridoma cell line is screened out; during the selection culture, half of the culture medium is generally changed every 2 to 3 days.
6. The method for preparing the drug-loaded human serum albumin combined monoclonal antibody according to claim 1, wherein the method comprises the following steps of: s9, the frozen stock solution is best precooled, and the operation is gentle and rapid; can be immediately reduced to 0 from room temperature during freezing placing in a refrigerator with ultralow temperature of-70 ℃, transferring into liquid nitrogen the next day; freezing can also be carried out by using a cell freezing device; the frozen cells are periodically resuscitated, and the activity of the cells and the stability of the secreted antibodies are checked, and the cells can be preserved for several years or more in liquid nitrogen; 2.6.2 method of cell resuscitation glass ampoule is carefully removed from liquid nitrogen, put in 37 ℃ water bath, thawing frozen cells in 1min, washing the cells twice with complete culture solution, then transferring into culture flask of feeder cells prepared on the first day, culturing in 37 ℃ 5% CO2 incubator, and detecting antibody activity when the cells form colonies.
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