CN114487436A - Kit for rapidly detecting whether hybridoma cells secrete antibodies - Google Patents

Kit for rapidly detecting whether hybridoma cells secrete antibodies Download PDF

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CN114487436A
CN114487436A CN202210055884.5A CN202210055884A CN114487436A CN 114487436 A CN114487436 A CN 114487436A CN 202210055884 A CN202210055884 A CN 202210055884A CN 114487436 A CN114487436 A CN 114487436A
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antibody
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hybridoma cells
hybridoma
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张洋
褚建达
张娅
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Zhejiang Zhengxi Biotechnology Co ltd
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    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract

A method for rapidly detecting antibody secretion from hybridoma cells, comprising: displaying the Fc segment of the antibody in the hybridoma cell in cytoplasm, and analyzing the Fc segment in cytoplasm to judge the specificity of the antibody and the antibody producing capacity of the cell. The method provided by the invention can visually detect the positive rate of the antibody-secreting cells in the total cell number in some antibodies expressed in cells and hybridoma cells. The antibody secretion capacity of each monoclonal cell and the nonspecific components on the surface of the hybridoma cells can be detected.

Description

Kit for rapidly detecting whether hybridoma cells secrete antibodies
Technical Field
The invention belongs to the field of cell biology, and particularly relates to a kit for rapidly detecting whether hybridoma cells secrete antibodies.
Background
An antibody is an immunoglobulin (Ig) that specifically binds an antigen and is hydrolyzed by proteolytic enzymes. Such as papain to 2 f (ab) and one Fc fragment. The F (ab) fragment of the antibody can bind antigen, while the Fc fragment functions as an Fc receptor binding, mediating opsonization (opsonization) or antibody-dependent cell-mediated cytotoxicity (ADCC). Since the Fc segment can be directly combined with enzyme or fluorescent dye to label the antibody, the Fc segment is the position of the rivet of the antibody on the fixing plate in ELISA experiments and the position for recognizing and combining the secondary antibody in immunoprecipitation, immunoblotting and immunohistochemistry.
The hybridoma is a hybrid cell which is screened in a limited culture medium after B cells with antibody secretion capacity and myeloma cells with unlimited proliferation capacity are fused in vitro, and has both antibody secretion capacity and unlimited in vitro proliferation capacity. During screening, hybridoma cell culture supernatant can be directly detected, and primary screening is completed. Generally, mouse ascites purified monoclonal antibody can be directly prepared by mouse hybridoma, but the yield is low. In addition, ascites cannot be prepared by preparing hybridomas from other animals such as rats, hamsters, rabbits, and the like. Therefore, the hybridoma cell strain is cultured in vitro, expanded and propagated, and then transferred into a fermentation tank for fermentation, and fermentation liquor is purified and collected to obtain a large number of pure antibiotic breakthrough points. The hybridoma cell strain capable of stably secreting the antibody is an important node in the process.
However, each hybridoma cell is the product of fusion of a B cell with a myeloma cell, and is unstable at passage during cell division and tends to lose the ability to produce antibodies. The lack of the Fc segment of the antibody, which results in the F (ab) segment being smaller and lacking the cross-linking function, makes it unable to bind to the antigen and precipitate, and is not captured by immune cells in vivo studies. Therefore, timely detection of the integrity of the antibody Fc segment of the hybridoma cells is an important prerequisite for ensuring stable antibody secretion of the hybridoma cells.
Disclosure of Invention
The invention aims to provide a kit for rapidly detecting whether a hybridoma cell secretes an antibody, which can lead the antibody generated by the hybridoma cell to be retained in the cell and not be secreted out of the cell by blocking the Golgi body of the hybridoma cell. The antibody in the hybridoma cell is released under the action of the cell membrane breaking agent, is specifically combined with a second antibody with fluorescence, and is subjected to fluorescence signal detection by using a flow technology, so that whether the hybridoma cell strain produces the antibody or not can be qualitatively analyzed, and the proportion of the cells with the antibody production capacity in the hybridoma cell to be detected, namely the strength of the antibody secretion capacity of the cell strain, can be quantitatively analyzed.
In one aspect, the invention provides a method for rapidly detecting antibody secretion from hybridoma cells.
The method comprises the following steps: displaying the Fc segment of the antibody in the hybridoma cell in cytoplasm, and analyzing the Fc segment in cytoplasm to judge the specificity of the antibody and the antibody producing capacity of the cell.
The method comprises the following steps:
s1, adding a protein transport inhibitor into the cell to be detected to enable the antibody secreted by the cell to be retained in the cell;
s2, performing membrane rupture and antibody fixation on the cells in the step S1;
s3, reacting the antibody in the step S2 with a secondary antibody;
s4, adding a buffer solution into the system after the reaction in the step S3, and detecting by a flow cytometer.
Preferably, the protein transport inhibitor in step S1 includes one or more of brefeldin a and monensin.
Preferably, the protein transport inhibitor in step S1 comprises brefeldin a; further preferably, the protein transport inhibitor is purchased from BD bioscience, cat # and manufacturer 555029.
The protease inhibitor is dissolved in RPMI-10% complete culture solution.
The ratio of the protease inhibitor to the RPMI-10% complete culture solution is 1: 10-1: 1000, parts by weight; preferably 1: 10.
specifically, the antibody in step S1 may be any antibody expressed by hybridoma cells, and the secondary antibody in step S3 may be any secondary antibody capable of binding to the antibody.
Preferably, the method for breaking the membrane in step S2 is to add cytofix/cytoperm.
Specifically, in step S3, the secondary antibody is one or more of a rat anti-mouse antibody, a goat anti-mouse antibody, and a rabbit anti-mouse antibody.
Preferably, the secondary antibody in step S3 is a goat anti-mouse antibody or a rabbit anti-mouse antibody.
Specifically, in step S3, the secondary antibody is a PE, FITC, APC or other fluorescent antibody.
Optionally, in step S3, the secondary antibody is fluorescent PE or FITC.
Alternatively, the cells to be detected in step S1 are one or more of CD22[10f.4.4.1], CD22[5E8.1.8], CD3[ SK7], and the secondary antibodies in step S3 are rat anti-mouse, goat anti-mouse and rabbit anti-mouse; preferably, the concentration of the secondary antibody is 0.5 mg/mL. Preferably, the secondary antibodies are goat anti-mouse IgG-PE and rabbit anti-mouse IgG-PE.
Preferably, the buffer in step S4 is Perm/Wash buffer.
Preferably, the Perm/Wash buffer in step S4 is used for washing first and resuspension second.
In another aspect, the invention provides a kit for rapidly detecting antibody secreted by hybridoma cells.
The kit comprises: protein transport inhibitor, cell membrane breaking agent, antibody fixing agent, secondary antibody reacting with the cell secretion antibody to be detected and buffer solution.
Preferably, the protein transport inhibitor is one or more of brefeldin A and monensin; more preferably bravadex a.
Preferably, the protein transport inhibitor is purchased from BD bioscience, cat # and manufacturer 555029.
Further preferably, the protease inhibitor is dissolved in RPMI-10% complete medium.
The ratio of the protease inhibitor to the RPMI-10% complete culture solution is 1: 10-1: 1000, parts by weight; preferably 1: 10.
preferably, the cell disrupting agent is cytofix/cytoperm.
Preferably, the buffer is Perm/Wash buffer.
The method for using the kit can be carried out by referring to the method for rapidly detecting the antibody secreted by the hybridoma cells.
Optionally, the cell to be detected is CD22[10f.4.4.1], CD22[5E8.1.8] or CD3[ SK7], and the secondary antibody in the step S3 is a rat anti-mouse antibody, a goat anti-mouse antibody and a rabbit anti-mouse antibody; preferably, the concentration of the secondary antibody is 0.5 mg/mL. Preferably, the secondary antibodies are goat anti-mouse IgG-PE and rabbit anti-mouse IgG-PE.
The invention has the beneficial effects that:
the strength of antibody secreted by cells can be directly detected by punching cell membranes, so that the positive rate of the antibody secreted by some antibodies expressed in the cells and hybridoma cells in the total cell number can be visually detected. The antibody secretion capacity of each monoclonal cell can be detected. The level of non-specific components on the surface of the hybridoma cells.
Drawings
FIG. 1 is a ratio of stained cells to total cells in a blank control; the left graph is a distribution graph of all cells in the cell suspension, the desired cells selected in the left graph are set as R2 gate, and the right graph is a distribution graph of cells with PE fluorescent signals in R2 gate.
FIG. 2 shows the proportion of cells stained with CD22[10F4.4.1] cells in total cells; the left panel is a distribution plot of all cells in the cell suspension, and the right panel is a distribution plot of cells with PE fluorescence signal in the R2 gate.
FIG. 3 is a proportion of cells stained with CD22[5E8.1.8] in total cells; the left panel is a distribution plot of all cells in the cell suspension, and the right panel is a distribution plot of cells with PE fluorescence signal in the R2 gate.
FIG. 4 shows the proportion of cells CD3[ SK7] in total cells; the left panel is a distribution plot of all cells in the cell suspension, and the right panel is a distribution plot of cells with PE fluorescence signal in the R2 gate.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
In the following examples, hybridoma cells were purchased from Shanghai owl Biotech GmbH.
Example 1A method for rapidly detecting antibody secretion from hybridoma cells
The cells to be tested were CD22[10f.4.4.1], expressing antibody types: mouse IgG.
Recovering cells, culturing for rejuvenation, counting, and collecting 4 × 106And centrifuging the cells to be detected for 5min at 1500rpm/min, adding 10mL of PBS buffer solution for washing, and centrifuging for 5min at 1500 rpm/min. After the centrifugation, 200. mu.L of complete culture medium containing protein transport inhibitor RPMI-10% was added.
The formulation of RPMI-10% complete medium (500mL) is shown in the following table:
Figure BDA0003476456340000041
the protein transport inhibitor is brefeldin A-containing protein transport inhibitor (555029/BD Biosciences), and has a formula of adding 600 μ L of brefeldin A-containing protein transport inhibitor into 6mL of RPMI-10% complete culture solution, blowing resuspended cells, sucking into round-bottom 96-well plate, placing in carbon dioxide incubator at 37 deg.C and 5% CO2Culturing at constant temperature for 3 hours to ensure that the antibody continuously secreted by the hybridoma cells is retained in the cells.
2. Taking out the cells to be detected from a carbon dioxide incubator, putting a round-bottom 96-well plate into a centrifuge, centrifuging at 2000rpm/min for 2 minutes, slightly pouring off the supernatant after the centrifugation is finished, leaving the cells to be detected at the bottom, adding 100 mu L of cytofix/cytoperm (51-2090KZ/BD Biosciences) into each cell hole, slightly blowing and beating, and reacting on ice for 30 minutes to break membranes of the hybridoma cells for fixation so as to facilitate the subsequent reaction of the antibody retained in the cells and a secondary antibody.
3. And (3) centrifuging the 96-well plate after the membrane rupture fixation is finished for 2min at 2000rpm, adding Perm/Wash buffer solution at 2000rpm/min after the centrifugation is finished, centrifuging for 2min, slightly pouring off the supernatant after the centrifugation is finished, leaving cells at the bottom of the 96-well plate with the round bottom, adding 50 mu L of rat anti-mouse with PE fluorescence into each well (the dilution ratio of the secondary antibody is 1: 50-1: 2000, the optimal range of 1:100, the subtype of the secondary antibody is IgG, the clone number is 4053, the concentration is 0.5mg/mL, the purchase source information is 405307, Biolgend), and reacting on ice for 15 min.
4. After the reaction is finished, 200 μ L of Perm/Wash buffer (51-2091KZ/BD Biosciences, the specific preparation method is that 1mL of Perm/Wash is added into 9mL of sterile water), the mixture is centrifuged at 2000rpm/min for 2 minutes, after 2 times of repetition, the supernatant is decanted, cells at the bottom of a 96-well round-bottom plate are left, 500 μ L of Perm/Wash buffer is added into each well, and after the resuspension, the mixture is transferred to a 1.5mL centrifuge tube to be used for signal detection by a flow cytometer.
The experimental results are as follows:
the cells stained in the blank accounted for 11.243% of the total cells (FIG. 1), and the cells stained with CD22[10F4.4.1] accounted for 44.552% of the total cells (FIG. 2).
Example 2
CD22[5E8.1.8] was tested according to the experimental method of example 1, and CD22[5E8.1.8] expressed Mouse IgG.
Cells stained CD22[5E8.1.8] accounted for 93.976% of the total cells (FIG. 3).
Example 3
CD3[ SK7] was tested and CD3[ SK7] expressed Mouse IgG1, kappa, according to the experimental method of example 1.
The cell CD3[ SK7] accounted for 0.352% of the total cells (fig. 4).
The above examples show that the cell positive rate of antibody secretion is highest in CD22[5E8.1.8], and then CD22[10F4.4.1], and no positive cell secretion is observed in CD3[ SK7 ].

Claims (10)

1. A method for rapidly detecting antibody secretion of hybridoma cells is characterized in that Fc segments of the antibodies in the hybridoma cells are shown in cytoplasm, and the Fc segments in the cytoplasm are analyzed to judge the specificity of the antibodies and the antibody production capacity of the cells.
2. The method of claim 1, comprising the steps of:
s1, adding a protein transport inhibitor into the cell to be detected to enable the antibody secreted by the cell to be retained in the cell;
s2, performing membrane rupture and antibody fixation on the cells in the step S1;
s3, reacting the antibody in the step S2 with a secondary antibody;
s4, adding a buffer solution into the system after the reaction in the step S3, and detecting by a flow cytometer.
3. The method according to claim 2, wherein the protein transport inhibitor in step S1 is one or more of brefeldin a and monensin.
4. The method of claim 2, wherein said protease inhibitor is dissolved in RPMI-10% complete medium; the ratio of the protease inhibitor to the RPMI-10% complete culture solution is 1: 10-1: 1000.
5. the method according to claim 2, wherein the cells to be detected in step S1 are one or more of CD22[10F.4.4.1], CD22[5E8.1.8], CD3[ SK7 ].
6. The method of claim 2, wherein the step S2 is performed by adding cytofix/cytoperm.
7. The method of claim 2, wherein the secondary antibody of step S3 is one or more of rat anti-mouse IgG, goat anti-mouse IgG, or rabbit anti-mouse IgG.
8. The method according to claim 2, wherein the buffer in step S4 is Perm/Wash buffer.
9. The method according to claim 8, wherein the Perm/Wash buffer of step S4 is used for washing first and for resuspension second.
10. A kit for rapidly detecting antibody secreted by hybridoma cells is characterized by comprising: a protein transport inhibitor, a cell membrane breaking agent, a second antibody reacting with a cell secretion antibody to be detected and a buffer solution.
CN202210055884.5A 2022-01-18 2022-01-18 Kit for rapidly detecting whether hybridoma cells secrete antibodies Pending CN114487436A (en)

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