CN111389053A - Immunoadsorbent, device and preparation method for purifying mouse monoclonal antibody in hybridoma cell culture solution - Google Patents
Immunoadsorbent, device and preparation method for purifying mouse monoclonal antibody in hybridoma cell culture solution Download PDFInfo
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Abstract
The invention provides an immunoadsorbent, a device and a preparation method for purifying a mouse monoclonal antibody in a hybridoma cell culture solution, wherein the immunoadsorbent comprises a solid phase carrier and an anti-mouse secondary antibody coupled on the solid phase carrier. When the solid phase carrier is nano particles, the nano particles coupled with the anti-mouse secondary antibody are specifically combined with the mouse monoclonal antibody, and then the purified mouse monoclonal antibody is obtained through desorption and separation. When the solid phase carrier is agarose gel and other materials, the solid phase carrier can be prepared into an immunoaffinity column device, and the device is used for purifying the mouse monoclonal antibody. The method solves the problem of bovine antibody interference in bovine serum when the mouse monoclonal antibody in the hybridoma cell culture solution is purified, avoids antibody waste caused by discarding the cell culture solution containing the mouse monoclonal antibody, improves the purity of the mouse monoclonal antibody, and increases the accuracy of subsequent experimental research on the mouse monoclonal antibody. Therefore, the invention plays an important role in the development of immune products.
Description
Technical Field
The invention belongs to the technical field of immunization, and particularly relates to an immunoadsorbent, a device and a preparation method for purifying a mouse monoclonal antibody in a hybridoma cell culture solution.
Background
Antibodies are immunoglobulins which can be specifically bound with antigens, and the unique biological activity of the antibodies plays an important role in disease diagnosis, immune control, biological detection and basic research. Antibodies are classified into polyclonal antibodies and monoclonal antibodies according to the number of epitopes recognized by the antibodies. Natural antigen molecules often contain a plurality of different epitopes, the antigen stimulates the immune system of the body to simultaneously activate a plurality of B cell clones, and the generated antibody contains a plurality of antibodies aiming at different epitopes, so the antibody is called polyclonal antibody. Plasma cells that are only directed against a single epitope are cloned and allowed to amplify in vitro secreted antibodies specific for the single epitope are referred to as monoclonal antibodies.
Monoclonal antibodies have the following advantages over polyclonal antibodies: (1) the hybridoma cells producing the antibody can survive and passage permanently in vitro, and the antibody with high specificity and high homogeneity can be produced continuously as long as the gene mutation of cell strains does not occur; (2)
the monoclonal antibody is suitable for immunological analysis methods characterized by labeled antibodies, such as IRMA and E L ISA, and (3) can be used for in vivo radioimmunoassay and immune-directed therapy due to the high specificity and single biological function of the monoclonal antibody.
Monoclonal antibodies are prepared mainly by fusing B lymphocytes of immunized mice with mouse myeloma cells to produce hybridoma cells, then culturing the hybridoma cells and selecting single cells capable of secreting antibodies with high sensitivity and good specificity, and culturing the single cells to propagate in large quantities to form clones of single hybridoma cells, also called cell lines, in the culture medium. Finally, the culture solution is discarded, and the cell line is frozen for standby. However, the discarded culture solution contains a large amount of monoclonal antibodies, and although the mass production level is not achieved, the antibodies contained in the culture solution can be purified for use, and particularly, the method is used for early experimental research, can obtain a small amount of antibodies with high purity before cell strains are frozen, and plays an important role in the rapid development of products in the later period.
At present, no technology is disclosed for extracting and purifying monoclonal antibodies in culture solution. The conventional antibody purification methods comprise an ammonium sulfate precipitation method, an ammonium sulfate caprylate precipitation method and a protein A or protein G purification method, and because the monoclonal cell culture solution must contain bovine serum, the conventional purification methods cannot distinguish bovine antibody interference in the bovine serum contained in the culture solution, so that the purity of the mouse monoclonal antibody extracted from the cell culture solution is reduced, and the accuracy of an experimental result is further influenced. At present, if a small amount of antibody is purified from a cell culture supernatant to carry out a preliminary test, cells are generally required to be cultured to a higher density and then replaced by a serum-free medium, but the cells die, and other operations such as cell cryopreservation, amplification culture and the like cannot be carried out any more, so that continuity is not provided.
Disclosure of Invention
In order to solve the problems of the prior art, the present invention aims to provide a device for purifying a mouse monoclonal antibody in a hybridoma cell culture solution and a preparation method thereof, which improve the purity of the mouse monoclonal antibody extracted from the culture solution, realize the full utilization of the antibody in the culture solution, and avoid the interference of the antibody component in the bovine serum.
In order to achieve the purpose, the invention provides the following technical scheme:
an immunoadsorbent for purifying a mouse monoclonal antibody in a hybridoma cell culture solution, which comprises a solid phase carrier and a mouse-resistant secondary antibody coupled to the solid phase carrier.
Further, the anti-mouse secondary antibody comprises a goat anti-mouse secondary antibody or a rabbit anti-mouse secondary antibody.
Further, the solid phase carrier is a nanoparticle.
Preferably, the nanoparticles include one or more of magnetic nanoparticles, gold nanoparticles, silver nanoparticles, gold-coated nanoparticles, silver-coated nanoparticles, nanorods, nanowires, nanostars, nano metal oxides, graphene oxide and quantum dots.
The method for purifying the mouse monoclonal antibody in the hybridoma cell culture solution by adopting the immunoadsorbent comprises the following steps:
(1) coupling an anti-murine secondary antibody to the surface of the nanoparticle;
(2) removing the unconjugated excess antibody;
(3) blocking the vacant sites on the surface of the nanoparticles;
(4) fully mixing the sealed nano particles with a hybridoma cell culture solution;
(5) separating the nanoparticles combined with the mouse monoclonal antibody after mixing;
(6) desorbing the mouse monoclonal antibody from the nanoparticles obtained in the step (5);
(7) separating the supernatant containing the mouse monoclonal antibody from the step (6), and discarding the nano particles to obtain a purified mouse monoclonal antibody;
when the nanoparticles only contain magnetic nanoparticles, the nanoparticles are magnetically separated by an external magnetic field;
when the nanoparticles do not contain magnetic nanoparticles or do not contain magnetic nanoparticles alone, the nanoparticles are separated by centrifugation.
Further, the solid phase carrier is one selected from agarose gel, cellulose, polyacrylamide gel, polyethylene gel, silica gel, chitosan or polystyrene-divinylbenzene resin, and each type of solid phase carrier comprises a modified derivative containing a functional group.
The device for purifying the mouse monoclonal antibody in the hybridoma cell culture solution prepared by the immunoadsorbent comprises a column tube and a solid-phase carrier filled in the column tube, wherein the upper end and the lower end of the solid-phase filler are respectively provided with a sieve plate.
Further, the preparation method of the device comprises the following steps:
(1) treating a solid phase carrier;
(2) coupling an anti-mouse secondary antibody to a solid phase carrier;
(3) blocking residual active groups on the solid phase carrier;
(4) removing unconjugated anti-mouse secondary antibody;
(5) and (5) column packing.
Has the advantages that: the invention provides an immunoadsorbent, a device and a preparation method for purifying a mouse monoclonal antibody in a hybridoma cell culture solution, and develops two methods for purifying the mouse monoclonal antibody in the hybridoma cell culture solution according to different types of solid phase carriers in the immunoadsorbent. When the solid phase carrier is nano particles, the nano particles coupled with the anti-mouse secondary antibody are specifically combined with the mouse monoclonal antibody, and then the purified mouse monoclonal antibody is obtained through desorption and separation. When the solid phase carrier is agarose gel and other materials, the solid phase carrier can be prepared into an immunoaffinity column device, and the device is used for purifying the mouse monoclonal antibody. The method solves the problem of bovine antibody interference in bovine serum when the mouse monoclonal antibody in the hybridoma cell culture solution is purified, avoids antibody waste caused by discarding the cell culture solution containing the mouse monoclonal antibody, improves the purity of the mouse monoclonal antibody, and increases the accuracy of subsequent experimental research on the mouse monoclonal antibody. Therefore, the invention plays an important role in the development of immune products.
Drawings
FIG. 1 is a schematic structural diagram of a device for purifying a mouse monoclonal antibody in a hybridoma cell culture solution according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
An immunoadsorbent for purifying a mouse monoclonal antibody in a hybridoma cell culture solution comprises a solid phase carrier and a mouse-resistant secondary antibody coupled to the solid phase carrier, wherein the mouse-resistant secondary antibody comprises a goat-anti-mouse secondary antibody or a rabbit-anti-mouse secondary antibody.
As an embodiment, the solid phase carrier is a nanoparticle, preferably, the nanoparticle includes one or a combination of several of magnetic nanoparticles, gold nanoparticles, silver nanoparticles, gold-coated nanoparticles, silver-coated nanoparticles, nanorods, nanowires, nanostars, nano metal oxides, graphene oxide, or quantum dots, and each nanoparticle also includes a nanoparticle obtained by functionally modifying the nanoparticle.
The method for purifying the mouse monoclonal antibody in the hybridoma cell culture solution by adopting the immunoadsorbent comprises the following steps:
(1) coupling an anti-murine secondary antibody to the surface of the nanoparticle;
(2) removing the unconjugated excess antibody;
(3) blocking the vacant sites on the surface of the nanoparticles;
(4) fully mixing the sealed nano particles with a hybridoma cell culture solution;
(5) separating the nanoparticles combined with the mouse monoclonal antibody after mixing;
(6) desorbing the mouse monoclonal antibody from the nanoparticles obtained in the step (5);
(7) separating the supernatant containing the mouse monoclonal antibody from the step (6), and discarding the nano particles to obtain a purified mouse monoclonal antibody;
when the nanoparticles only contain magnetic nanoparticles, the nanoparticles are magnetically separated by an external magnetic field;
when the nanoparticles do not contain magnetic nanoparticles or do not contain magnetic nanoparticles alone, the nanoparticles are separated by centrifugation.
In another embodiment, the solid phase carrier is one selected from agarose gel, cellulose, polyacrylamide gel, polyvinyl gel, silica gel, chitosan or polystyrene-divinylbenzene resin, and each type of solid phase carrier comprises a modified derivative containing a functional group.
The device for preparing and purifying the mouse monoclonal antibody in the hybridoma cell culture solution by using the immunoadsorbent comprises a column tube and a solid phase carrier filled in the column tube, wherein the upper end and the lower end of the solid phase filler are respectively provided with a sieve plate.
The preparation method of the device comprises the following steps:
(1) treating a solid phase carrier;
(2) coupling an anti-mouse secondary antibody to a solid phase carrier;
(3) blocking residual active groups on the solid phase carrier;
(4) removing unconjugated anti-mouse secondary antibody;
(5) and (5) column packing.
The hybridoma cell culture fluids used in the following examples and comparative examples were obtained by the following preparation methods:
fusing a mouse spleen cell for immunizing aflatoxin and a mouse myeloma cell to prepare a monoclonal antibody, detecting a positive cell hole by using an indirect competitive enzyme-linked immunosorbent assay, screening a cell hole with a good inhibition effect, and taking out a cell culture solution in the cell hole with the good inhibition effect as a raw material for purifying the mouse monoclonal antibody.
Example 1 with Fe3O4Preparation of immunoadsorbent from magnetic nanoparticles for mouse monoclonal antibody in hybridoma cell culture solution
The cell culture broth was dialyzed beforehand against PBS buffer pH 7.40.01M.
Commercial 50mg of 150nm carboxylated Fe with a particle size of 100-3O4Dispersing magnetic nanoparticles in MES buffer solution with pH of 6.0-6.5, performing ultrasonic dispersion for 10min, adding 5mg NHS under oscillation condition, performing oscillation reaction for 20min at room temperature, adding 10 mg EDC, performing oscillation reaction for 1h to activate the magnetic nanoparticles, removing supernatant through magnetic separation, washing the magnetic nanoparticles through magnetic separation by PBS buffer solution with pH of 7.40.01M, performing resuspension on the activated magnetic nanoparticles by 1M L PBS buffer solution, adding 1mg goat-anti-mouse secondary antibody, performing oscillation reaction for 2h at room temperature, removing supernatant through magnetic separation, adding OVA with pH of 1M L1 mg/M L, performing dispersion on the magnetic nanoparticles, performing oscillation reaction for 2h, washing the magnetic nanoparticles 3 times by PBS buffer solution, adding dialyzed cell culture solution into the washed magnetic nanoparticles, performing oscillation mixing for 30min at 37 ℃, collecting the magnetic nanoparticles through magnetic separation, removing supernatant, adding 1M L methanol solution into the magnetic nanoparticles, performing oscillation mixing for 10min, removing the supernatant through PBS, performing nitrogen blowing analysis to obtain purified antibody M7.40.01.
EXAMPLE 2 mouse monoclonal antibodies in hybridoma cell culture fluid with immunoadsorbent prepared on agarose gel
(1) The goat anti-mouse secondary antibody was placed in advance in 0.1 mol/L NaHCO solution containing 0.5 mol/L NaCl at pH8.33Dialyzing in a coupling buffer solution, repeatedly washing 0.5g of cyanogen bromide activated agarose dry powder Sepharose4B with 1mM hydrochloric acid for more than 15min to swell the Sepharose, quickly washing the Sepharose in a sand core funnel by using the coupling buffer solution to remove impurities, dissolving agarose in 5m L of the coupling buffer solution, adding 0.8mg of goat-anti-mouse secondary antibody, oscillating at room temperature to fully react for 1h and uniformly mixing, and washing the uncoupled goat-anti-mouse secondary antibody by using more than 5 times of the volume of the coupling buffer solution to obtain the immunoadsorbent;
(2) blocking active groups, namely transferring the immunoadsorbent into 0.1 mol/L Tris-HCl buffer solution with the volume of 5 times and the pH value of 8.0 and containing 0.5 mol/L NaCl, and keeping for 2 hours to block redundant active groups in the agarose gel;
(3) washing the immunoadsorbent obtained in the step (2) with 5 times of volume of 0.1 mol/L acetic acid buffer solution containing 0.5 mol/L NaCl at pH4.0 and 5 times of volume of 0.1 mol/L Tris-HCl buffer solution containing 0.5 mol/L NaCl at pH8.0, wherein the washing process is repeated three times and then washed twice with 5 times of volume of PBS;
(4) and (3) antiseptic treatment: soaking the immunoadsorbent prepared in the step (3) in PBS (phosphate buffer solution) containing 1/10000 sodium azide, draining off redundant solution, and then putting into a packaging bag or a bottle for sealed storage at 4 ℃;
(5) filling the immunoadsorbent obtained in the step (3) or (4) into a 1m L solid phase extraction hollow column tube, compacting the gel by PBS under certain negative pressure, and balancing by 5% methanol for later use to prepare the immunoaffinity column for purifying the mouse monoclonal antibody in the hybridoma cell culture solution;
(6) and (3) storage: the prepared immunoaffinity column was stored in a refrigerator at 4 ℃.
Dialyzing the cell culture solution by PBS containing 10% methanol in advance, washing the prepared immunoaffinity column by 15m L pure water, then passing the dialyzed cell culture solution through the column, washing the column by 15m L pure water to remove the unbound solution, finally eluting by 1m L methanol solution, collecting the eluent, and detecting the content of the mouse monoclonal antibody in the eluent by liquid chromatography.
Whether the purified antibodies of the example 1 and the example 2 contain the bovine antibody is identified by adopting an E L ISA method, a cell culture solution dialyzed by PBS only is taken as a comparative example, and a rabbit anti-bovine secondary antibody marked by HRP is taken as a detection antibody.
(1) Coating, the initial concentration of total protein in example 1, example 2 and comparative example was diluted 10-fold in 100 μ g/M L gradient with 0.05M pH 9.6 carbonate buffer at 100 μ L per well and reacted at 37 ℃ for 2h, blank control wells were coated with 5% OVA;
(2) washing: the plate solution was decanted and washed 3 times with wash solution (PBST), 3min each time;
(3) sealing, adding 200 mu L/hole sealing solution (5% OVA) after patting dry, reacting for 2h at 37 ℃, washing and drying for later use;
(4) adding sample, adding HRP-rabbit anti-bovine IgG diluted at a ratio of 1:3000 and with a concentration of 100 mu L/hole, and reacting at 37 ℃ for 30 min;
(5) color development, the enzyme label plate is taken out and fully washed, then TMB color development liquid of 100 mu L is added into each hole, and the reaction is carried out at 37 ℃ in the dark
15min；
(6) Stop and assay 50. mu. L stop solution was added to each well to stop the reaction, and then OD450nm values were measured for each well using a plate reader, and the results are shown in Table 1.
As can be seen from Table 1, the color value of the antibody purified in example 1 and example 2 after being detected by E L ISA is close to that of the blank control hole, while the color value of the comparative example is deepened along with the increase of the concentration of the coated antibody, thus the method of the invention can effectively solve the interference of the cattle antibody existing in the cell culture solution on the purification of the mouse monoclonal antibody, realize the purification of the mouse monoclonal antibody in the cell culture solution, and improve the utilization rate of the monoclonal antibody in the cell culture solution, wherein the cattle antibody does not exist in example 1 and example 2, and the cattle antibody exists in the comparative example, namely unpurified cell culture solution.
TABLE 1
Claims (8)
1. An immunoadsorbent for purifying a mouse monoclonal antibody in a hybridoma cell culture solution, which is characterized by comprising a solid phase carrier and an anti-mouse secondary antibody coupled to the solid phase carrier.
2. The immunoadsorbent of claim 1, wherein said anti-mouse secondary antibody comprises goat anti-mouse secondary antibody or rabbit anti-mouse secondary antibody.
3. The immunoadsorbent of claim 1, wherein said solid support is a nanoparticle.
4. The immunoadsorbent of claim 3, wherein the nanoparticles comprise one or more of magnetic nanoparticles, gold nanoparticles, silver nanoparticles, gold-coated nanoparticles, silver-coated nanoparticles, nanorods, nanowires, nanostars, metal oxides, graphene oxide, and quantum dots.
5. A method for purifying murine mAb in hybridoma cell culture using the immunoadsorbent of claim 3 or 4, comprising the steps of:
(1) coupling an anti-murine secondary antibody to the surface of the nanoparticle;
(2) removing the unconjugated excess antibody;
(3) blocking the vacant sites on the surface of the nanoparticles;
(4) fully mixing the sealed nano particles with a hybridoma cell culture solution;
(5) separating the nanoparticles combined with the mouse monoclonal antibody after mixing;
(6) desorbing the mouse monoclonal antibody from the nanoparticles obtained in the step (5);
(7) separating the supernatant containing the mouse monoclonal antibody from the step (6), and discarding the nano particles to obtain a purified mouse monoclonal antibody;
when the nanoparticles only contain magnetic nanoparticles, the nanoparticles are magnetically separated by an external magnetic field;
when the nanoparticles do not contain magnetic nanoparticles or do not contain magnetic nanoparticles alone, the nanoparticles are separated by centrifugation.
6. The immunoadsorbent of claim 1, wherein said solid support is selected from the group consisting of agarose gel, cellulose, polyacrylamide gel, polyethylene gel, silica gel, chitosan, and polystyrene-divinylbenzene, and each type of solid support comprises a modified functional group-containing derivative.
7. The device for purifying the mouse monoclonal antibody in the hybridoma cell culture solution prepared by the immunoadsorbent of claim 6, which is characterized by comprising a column tube and a solid phase carrier filled in the column tube, wherein the upper end and the lower end of the solid phase filler are provided with sieve plates.
8. A method for preparing the device of claim 7, comprising the steps of:
(1) treating a solid phase carrier;
(2) coupling an anti-mouse secondary antibody to a solid phase carrier;
(3) blocking residual active groups on the solid phase carrier;
(4) removing unconjugated anti-mouse secondary antibody;
(5) and (5) column packing.
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| CN113457214A (en) * | 2021-06-08 | 2021-10-01 | 上海海路生物技术有限公司 | Solid phase extraction system and method combining syringe type gravity column and solid phase extractor |
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