CN113943712A - Electrofusion buffer solution, preparation method thereof and electrofusion method - Google Patents
Electrofusion buffer solution, preparation method thereof and electrofusion method Download PDFInfo
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- 239000007853 buffer solution Substances 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title description 6
- 210000004027 cell Anatomy 0.000 claims abstract description 41
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 21
- 239000000872 buffer Substances 0.000 claims description 38
- 239000000243 solution Substances 0.000 claims description 28
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 26
- 229940098773 bovine serum albumin Drugs 0.000 claims description 26
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 23
- 239000011654 magnesium acetate Substances 0.000 claims description 23
- 229940069446 magnesium acetate Drugs 0.000 claims description 23
- 235000011285 magnesium acetate Nutrition 0.000 claims description 23
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 claims description 22
- 239000001639 calcium acetate Substances 0.000 claims description 22
- 229960005147 calcium acetate Drugs 0.000 claims description 22
- 235000011092 calcium acetate Nutrition 0.000 claims description 22
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 21
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 21
- 229960002920 sorbitol Drugs 0.000 claims description 21
- 239000006285 cell suspension Substances 0.000 claims description 11
- 230000005684 electric field Effects 0.000 claims description 11
- 230000004927 fusion Effects 0.000 claims description 8
- 230000009471 action Effects 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 6
- 210000004989 spleen cell Anatomy 0.000 claims description 5
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 4
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 230000007910 cell fusion Effects 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 13
- 238000002474 experimental method Methods 0.000 abstract description 11
- 230000004083 survival effect Effects 0.000 abstract description 5
- 239000011324 bead Substances 0.000 abstract description 3
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 7
- 239000012498 ultrapure water Substances 0.000 description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 241001146209 Curio rowleyanus Species 0.000 description 1
- 101001010621 Homo sapiens Interleukin-21 Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 239000007785 strong electrolyte Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
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- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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Abstract
The invention provides an electrofusion buffer solution and a method for electrofusion of cells, which can greatly improve the cell fusion efficiency and the cell survival rate of a hybridoma technology. The invention optimizes the proportion of the electrofusion buffer solution, the buffer solution not only has low conductivity, but also solves the problems of interference when the cell beads are arranged and the heat effect of the cell fusion experiment, improves the clone formation rate and reduces the experiment cost.
Description
Technical Field
The invention belongs to the technical field of hybridoma cell production and application, and particularly relates to a buffer solution with low conductivity and a method for electrically fusing cells.
Background
The hybridoma technology is also called monoclonal antibody technology, and mainly comprises the steps of preparing a monoclonal antibody specific to an antigen, fusing B lymphocytes of an immunized animal (usually spleen cells of the immunized animal) with myeloma cells to obtain hybridoma cells capable of generating antibodies and continuously proliferating, and further producing a large amount of monoclonal antibodies. Although the hybridoma technology is an important basis for producing monoclonal antibodies, the experimental steps of the technology are complicated, the period is long, and factors influencing the success of the experiment are numerous, wherein the hybridoma technology is a manufacturing process. The hybridoma generated at the beginning of fusion is easily influenced by small environmental changes and extremely easily fails; even if the hybridoma cells grow well, the whole experiment may fail because the hybridoma cells cannot secrete specific antibodies, and the experiment cost is easily increased.
In order to improve cloning efficiency, improve success rate of obtaining specific antibodies and obtain high yield of antibodies, the prior art adjusts the culture medium of hybridoma cells, and CN103865880A discloses a culture medium and a preparation method thereof, wherein the culture medium comprises 1640 complete culture medium and recombinant human IL-21, which can promote growth of newly generated hybridoma cells and enhance the ability of the hybridoma cells to secrete specific antibodies. However, in such a method, the culture environment is modified after the production of the hybridoma cells, and if the number of the hybridoma cells obtained by cell fusion is small or the production efficiency is poor, the above experiment may fail. Therefore, how to improve the cell fusion efficiency and cell survival rate can be a big key.
Disclosure of Invention
The invention aims to improve the cell fusion efficiency and the cell survival rate and optimize the cell electrofusion experimental conditions, and provides a formula of an electrofusion buffer solution and an electrofusion method. The buffer solution not only has low conductivity, but also solves the problem of damage to object cells caused by strong conductivity, has a certain protection effect on cytoplasm, and reduces the interference to the arrangement of cell beads and the heat effect of cell fusion experiments to the maximum extent.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides an electrofusion buffer solution, which is characterized by comprising the following components: 0.2 to 0.3M D-sorbitol, 0.1 to 0.3mM magnesium acetate, 0.1 to 0.2mM calcium acetate and 0.5 to 2mg/mL bovine serum albumin.
Preferably, the electrofusion buffer according to the present invention is characterized by comprising the following components: 0.3M D-sorbitol, 0.1mM magnesium acetate, 0.1mM calcium acetate and 1 mg/mL bovine serum albumin.
In one embodiment, the invention provides an electrofusion buffer, which is characterized in that the electrofusion buffer comprises D-sorbitol, magnesium acetate, calcium acetate and bovine serum albumin, wherein the mass ratio of the D-sorbitol to the magnesium acetate to the calcium acetate to the bovine serum albumin is 36.4-54.6: 0.0214 to 0.0642: 0.0158-0.0316: 0.5 to 2.
More preferably, the electrofusion buffer of any one of the present invention has a pH of 6.8 to 7.2.
More preferably, the electrofusion buffer according to any of the present invention is used in hybridoma technology or monoclonal antibody technology.
In one embodiment, the present invention provides a method for preparing any one of the electrofusion buffers described above, including the sub-step of:
(1) dispersing D-sorbitol into pure water to obtain a first solution;
(2) adding bovine serum albumin into the first solution to obtain a second solution;
(3) adding magnesium acetate into the second solution to obtain a third solution;
(4) and adding calcium acetate into the third solution, and filtering to obtain the electrofusion buffer solution.
Preferably, the preparation method is characterized in that after the electrofusion buffer solution is fully dissolved and uniformly mixed, the pH value is adjusted to 6.8-7.2.
In one embodiment, the present invention provides a method for electrofusion of cells, characterized in that said method comprises the sub-step of:
(1) mixing myeloma cells and spleen cells according to the quantitative ratio of 1: 2-1: 5;
(2) resuspending the cells of step (1) in the electrofusion buffer of any one of claims 1-3;
(3) adding the cell suspension obtained in the step (2) into a fusion chamber of an electrofusion instrument;
(4) applying an alternating voltage;
(5) applying two direct current pulses, and applying an alternating current voltage again after the next direct current pulse;
(6) and transferring the cell suspension after electrofusion to HAT culture medium, and adding the cell suspension into a 96-well plate paved with feeder cells for culture screening.
Preferably, the method of the present invention is characterized in that the cell suspension of the step (2) is 1X 107Individual cells/mL.
Preferably, the method of the present invention is characterized in that the ac voltage parameters of step (4) are: the alternating current electric field is 30V and 50V, the frequency of the alternating current electric field is 2 MHz, and the duration of action is 20 seconds.
Preferably, the method of the present invention is characterized in that the two dc electrical pulse parameters of step (5) are: the direct current pulse voltage is 400V and 800V, and the action time is 0.5 second.
Preferably, the method of the present invention is characterized in that the ac voltage parameters of step (5) are: the alternating current electric field is 50V, the frequency of the alternating current electric field is 0.8 MHz, and the duration of action is 7 seconds.
Compared with the prior art, the electrofusion buffer solution has the beneficial effects that:
(1) greatly improves the cell fusion rate or the clone formation rate, and is beneficial to the selection of the hybridoma.
(2) Reduce the interference when arranging the string of beads of cell and the heat effect of cell fusion experiment, reduce the damage to the cell, promote the cell survival rate.
(3) The success rate of the hybridoma technology experiment is improved, and the cost is reduced.
(4) The cost is far lower than that of the commercial electrofusion buffer solution product.
Detailed Description
The present invention is further described with reference to specific examples, which are not intended to limit the scope of the present invention. It is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It is intended that all such alterations and advantages be included in the invention, which occur to those skilled in the art, be considered as within the spirit and scope of the inventive concept, and that all such modifications and advantages be considered as within the scope of the appended claims and any equivalents thereof.
All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In other instances, certain terms used herein will have their meanings as set forth in the specification. The experimental procedures in the following examples, in which specific conditions are not specified, are all common knowledge and general knowledge of those skilled in the art, or conditions recommended by the manufacturer. All materials and reagents used in the examples are commercially available products unless otherwise specified. The embodiments and features of the embodiments in the present application may be combined with each other.
The electrofusion buffer is a main factor influencing electrofusion, and both osmotic pressure and components influence the effect of electrofusion. The electrofusion can be directly observed under an inverted microscope, so that the fusion conditions can be controlled and selected purposefully.
The invention provides a buffer solution in electrofusion, which has the characteristics that:
(1) the dissociation is sufficient, the damage of strong conductivity to the object cells is solved, the interference to the arrangement of the cell beads is greatly reduced, and the heat effect of the cell fusion experiment is reduced.
(2) Bovine Serum Albumin (BSA) provides nutrients for cells, has the function of protecting cytoplasm, and improves the efficiency and the survival rate of the cells during cell fusion.
(3) Improve the clone formation rate and is beneficial to the selection of hybridoma.
The present invention will be described in detail with reference to examples.
Example 1: preparation of high and low concentration electrofusion buffer solution and its cell fusion efficiency
The high-concentration and low-concentration electrofusion buffer solution is prepared as follows:
A. low concentration protocol: 0.2M D-sorbitol, 0.1mM magnesium acetate, 0.1mM calcium acetate, 0.5 mg/mL bovine serum albumin
Step 1: 18.2g D-sorbitol was weighed and dissolved in 400mL of ultrapure water.
Step 2: 250mg of Bovine Serum Albumin (BSA) (0.5 mg/mL) was weighed and dissolved in the buffer of step 1.
And step 3: 10.7mg of magnesium acetate was weighed and dissolved in the buffer of step 2.
And 4, step 4: 7.9mg of calcium acetate was weighed and dissolved in the buffer of step 3.
And 5: after the solution is sufficiently dissolved, the pH value is measured to be 6.8-7.2, the volume is determined to be 500mL, the solution is filtered by a 0.2 mu m filter, and the solution is stored at 4 ℃ for standby.
B. High concentration protocol: 0.3M D-sorbitol, 0.3mM magnesium acetate, 0.2mM calcium acetate, 2mg/mL bovine serum albumin
Step 1: 27.3g D-sorbitol was weighed and dissolved in 400mL of ultrapure water.
Step 2: 1000mg of Bovine Serum Albumin (BSA) (2 mg/mL) was weighed and dissolved in the buffer of step 1.
And step 3: 32.1mg of magnesium acetate was weighed and dissolved in the buffer of step 2.
And 4, step 4: 15.8mg of calcium acetate was weighed and dissolved in the buffer of step 3.
And 5: after the solution is sufficiently dissolved, the pH value is measured to be 6.8-7.2, the volume is determined to be 500mL, the solution is filtered by a 0.2 mu m filter, and the solution is stored at 4 ℃ for standby.
Experimental results and data, as shown in table 2:
in summary, the cell fusion rate after the low-concentration and high-concentration electrofusion buffer solution is fused is 0.56-0.61%, and the technical scheme of the invention needs to further optimize the formula and test the effect.
Example 2: preparation of four electrofusion buffers
The invention is debugged in the high concentration and low concentration range and compared with the current commonly used electric melting liquid.
The electric melting liquid configurations of the four schemes are as follows:
the first scheme is as follows: 0.3M mannitol, 0.1mM calcium chloride, 0.1mM magnesium chloride
Step 1: 27.3258g of mannitol was weighed out and dissolved in 450mL of ultrapure water.
Step 2: 406mg of magnesium chloride was weighed and dissolved in 20mL of ultrapure water.
And step 3: 438mg of calcium chloride was weighed and dissolved in the buffer of step 2.
And 4, step 4: and (3) sucking 500 mu L of the buffer solution prepared in the step (3), adding the buffer solution into the solution in the step (1), fully and uniformly mixing, metering the volume to 500mL, filtering by using a 0.2 mu m filter, and storing at 4 ℃ for later use.
The effect is as follows: calcium chloride and magnesium chloride belong to strong electrolytes, the alternating current pulse in a buffer solution with strong conductivity and high conductivity can damage the cell activity, and when the resistance is 104 omega/cm, the alternating current heat rises, so that the cell stringing arrangement and the cell-cell close contact are prevented, and the cell fusion and development are adversely affected.
Scheme II: 0.3M D-sorbitol, 0.5mM magnesium acetate, 0.1mM calcium acetate
Step 1: 27.3g D-sorbitol was weighed out and dissolved in 400mL of ultrapure water.
Step 2: 53.5mg of magnesium acetate was weighed and dissolved in the buffer of step 1.
And step 3: 7.9mg of calcium acetate was weighed and dissolved in the buffer of step 2.
And 4, step 4: after the solution is sufficiently dissolved, the pH value is measured to be 6.8-7.2, the volume is determined to be 500mL, the solution is filtered by a 0.2 mu m filter, and the solution is stored at 4 ℃ for standby.
The effect is as follows: compared with the first scheme, the calcium acetate and the magnesium acetate have relatively weak conductivity, and can reduce the damage to cells.
The third scheme is as follows: 0.3M D-sorbitol, 0.1mM magnesium acetate, 0.1mM calcium acetate, 1 mg/mL bovine serum albumin
Step 1: 27.3g D-sorbitol was weighed and dissolved in 400mL of ultrapure water.
Step 2: 500mg of Bovine Serum Albumin (BSA) (1 mg/mL) was weighed and dissolved in the buffer of step 1.
And step 3: 10.7mg of magnesium acetate was weighed and dissolved in the buffer of step 2.
And 4, step 4: 7.9mg of calcium acetate was weighed and dissolved in the buffer of step 3.
And 5: after the solution is sufficiently dissolved, the pH value is measured to be 6.8-7.2, the volume is determined to be 500mL, the solution is filtered by a 0.2 mu m filter, and the solution is stored at 4 ℃ for standby.
The effect is as follows: compared with the second scheme, the addition of the bovine serum albumin can not only provide nutrients for cells, but also has a certain protection effect on cytoplasm.
And the scheme is as follows: 3M D-sorbitol, 0.5mM magnesium acetate, 0.1mM calcium acetate, 1 mg/mL bovine serum albumin
Step 1: 27.3g D-sorbitol was weighed out and dissolved in 400mL of ultrapure water.
Step 2: 500mg of Bovine Serum Albumin (BSA) (1 mg/mL) was weighed and dissolved in the buffer of step 1.
And step 3: 53.5mg of magnesium acetate was weighed and dissolved in the buffer of step 2.
And 4, step 4: 7.9mg of calcium acetate was weighed out and dissolved in the buffer of step 3.
And 5: after the solution is sufficiently dissolved, the pH value is measured to be 6.8-7.2, the volume is determined to be 500mL, the solution is filtered by a 0.2 mu m filter, and the solution is stored at 4 ℃ for standby.
The effect is as follows: compared with the third scheme, the magnesium acetate has higher concentration and higher conductivity, and has certain damage to cells.
Example 3: comparison of cell fusion efficiency of four electrofusion buffers
Performing electrofusion by the following steps:
1. after the spleen cells of SPF-grade mice are separated, mouse myeloma cells Sp2/0 cells (Sp 2/0-Ag14, ATCC number: CRL-1581) and the spleen cells are mixed in a ratio of 1: 2-1: 5 respectively.
2. After the cell suspension is washed twice by the four electrofusion buffers, the cells are resuspended by the four electrofusion buffers for fusion.
3. An electrofusion apparatus (BTX, USA, model: ECM 2001) was equipped with 2 mL of electrodes, and the number of cells was adjusted to 1X 10 using the four electrofusion buffers of example 17And (4) adding the mixed cell suspension into the fusion chamber.
4. The arrangement of cells was observed by microscope by adjusting the alternating voltage, i.e., the alternating electric field (AC) intensity was designed to be 30V and 50V, the frequency of the alternating electric field was 2 MHz, and the duration of the alternating electric field was 20 seconds.
5. Two direct current pulses are used, the direct current pulse voltage (DC) is respectively designed to be 400V and 800V, and the action time of the direct current pulse voltage is 0.5 second. After the last pulse, an alternating voltage of 50V, 0.8 MHz, was used for 7 seconds.
6. After fusion, the cell suspension was transferred from the fusion chamber to a preheated HAT-containing medium (Sigma, cat # H0262-10 VL) to adjust the cell count to 4X 105100 μ L/mL of cells were plated in 96-well plates plated with primary cells of the peritoneal fluid of SPF mice as feeder cells at 37 ℃ with 5% CO2Culturing in medium.
7. After 5 days of culture, the number of colonies of hybridoma cells was recorded; and after culturing for 7-8 days, taking cell culture supernatant to screen positive hybridoma cells.
Experimental results and data, as shown in table 4:
in conclusion, the cell fusion rate of the electrofusion buffer prepared by the third scheme is obviously higher than that of the electrofusion buffer prepared by other formulas.
A preferred formulation of the invention is 0.3M D-sorbitol, 0.1mM magnesium acetate, 0.1mM calcium acetate, 1 mg/mL bovine serum albumin.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All documents referred to herein are incorporated by reference in their entirety. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art without departing from the spirit and principles of the invention, and all such modifications and equivalents fall within the scope of the claims of the invention.
Claims (12)
1. An electrofusion buffer comprising the following components: 0.2 to 0.3M D-sorbitol, 0.1 to 0.3mM magnesium acetate, 0.1 to 0.2mM calcium acetate and 0.5 to 2mg/mL bovine serum albumin.
2. The electrofusion buffer of claim 1 comprising the following components: 0.3M D-sorbitol, 0.1mM magnesium acetate, 0.1mM calcium acetate and 1 mg/mL bovine serum albumin.
3. The electrofusion buffer solution is characterized by comprising D-sorbitol, magnesium acetate, calcium acetate and bovine serum albumin, wherein the mass ratio of the D-sorbitol to the magnesium acetate to the calcium acetate to the bovine serum albumin is 36.4-54.6: 0.0214 to 0.0642: 0.0158-0.0316: 0.5 to 2.
4. The electrofusion buffer according to any of claims 1-3 wherein the electrofusion buffer has a pH of 6.8 to 7.2.
5. Use of an electrofusion buffer according to any of claims 1-3 in hybridoma technology or monoclonal antibody technology.
6. The method for preparing an electrofusion buffer according to any of the claims 1-3, characterised in that it comprises the sub-step of:
(1) dispersing D-sorbitol into pure water to obtain a first solution;
(2) adding bovine serum albumin into the first solution to obtain a second solution;
(3) adding magnesium acetate into the second solution to obtain a third solution;
(4) and adding calcium acetate into the third solution, and filtering to obtain the electrofusion buffer solution.
7. The method according to claim 6, wherein the electrofusion buffer is dissolved sufficiently and mixed uniformly, and then the pH value is adjusted to 6.8-7.2.
8. A method for electrofusion of cells, said method comprising the sub-step of:
(1) mixing myeloma cells and spleen cells according to the quantitative ratio of 1: 2-1: 5;
(2) resuspending the cells of step (1) in the electrofusion buffer of any one of claims 1-3;
(3) adding the cell suspension obtained in the step (2) into a fusion chamber of an electrofusion instrument;
(4) applying an alternating voltage;
(5) applying two direct current pulses, and applying an alternating current voltage again after the next direct current pulse;
(6) and transferring the cell suspension after electrofusion to HAT culture medium, and adding the cell suspension into a 96-well plate paved with feeder cells for culture screening.
9. The method according to claim 8, wherein the cell suspension of step (2) is 1X 107Individual cells/mL.
10. The method according to claim 8, wherein the alternating voltage parameters of step (4) are: the alternating current electric field is 30V and 50V, the frequency of the alternating current electric field is 2 MHz, and the duration of action is 20 seconds.
11. The method according to claim 8, wherein the two DC electrical pulse parameters of step (5) are: the direct current pulse voltage is 400V and 800V, and the action time is 0.5 second.
12. The method according to claim 8, wherein the alternating voltage parameters of step (5) are: the alternating current electric field is 50V, the frequency of the alternating current electric field is 0.8 MHz, and the duration of action is 7 seconds.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999005266A2 (en) * | 1997-07-26 | 1999-02-04 | Wisconsin Alumni Research Foundation | Trans-species nuclear transfer |
| US20030082163A1 (en) * | 2001-10-26 | 2003-05-01 | Suyu Shu | Fused cells, methods of forming same, and therapies utilizing same |
| US20060037086A1 (en) * | 2003-04-09 | 2006-02-16 | Schatten Gerald P | Methods for correcting mitotic spindle defects and optimizing preimplantation embryonic developmental rates associated with somatic cell nuclear transfer in animals |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999005266A2 (en) * | 1997-07-26 | 1999-02-04 | Wisconsin Alumni Research Foundation | Trans-species nuclear transfer |
| US20030082163A1 (en) * | 2001-10-26 | 2003-05-01 | Suyu Shu | Fused cells, methods of forming same, and therapies utilizing same |
| US20060037086A1 (en) * | 2003-04-09 | 2006-02-16 | Schatten Gerald P | Methods for correcting mitotic spindle defects and optimizing preimplantation embryonic developmental rates associated with somatic cell nuclear transfer in animals |
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