Kit for rapidly detecting whether hybridoma cells secrete antibodies or not

A hybridoma cell and cell secretion technology, which is applied in the field of kits for rapid detection of whether hybridoma cells secrete antibodies, can solve the problems of unstable passage, lack of cross-linking function, and loss of antibody production.

Pending Publication Date: 2022-05-13
ZHEJIANG ZHENGXI BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, each hybridoma cell is the product of the fusion of B cells and myeloma cells, which is unstable during cell division and prone to lose the ability to produce antibodies
The absence of the Fc segment of

Method used

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  • Kit for rapidly detecting whether hybridoma cells secrete antibodies or not
  • Kit for rapidly detecting whether hybridoma cells secrete antibodies or not
  • Kit for rapidly detecting whether hybridoma cells secrete antibodies or not

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 A method for rapidly detecting antibodies secreted by hybridoma cells

[0045] The cells to be detected are CD22[10F.4.4.1], and the type of antibody expressed: Mouse IgG.

[0046] Resuscitated cells, counted after rejuvenation, and took 4×10 6 Each cell to be tested was centrifuged at 1500rpm / min for 5min, then washed with 10mL PBS buffer, and centrifuged at 1500rpm / min for 5min. After centrifugation, 200 μL of RPMI-10% complete culture solution containing protein transport inhibitor was added.

[0047] RPMI-10% complete culture solution (500mL) formulation is shown in the following table:

[0048]

[0049]The protein transport inhibitor is a protein transport inhibitor containing brefedex A (555029 / BD Biosciences), and the formula is 6 mL of RPMI-10% complete culture solution, adding 600 μL of a protein transport inhibitor containing brefexedin A, resuspended cells by pipetting, Pipet into a round-bottom 96-well plate in a carbon dioxide incubator at 3...

Embodiment 2

[0056] Referring to the experimental method of Example 1, CD22[5E8.1.8] was detected, and CD22[5E8.1.8] expressed MouseIgG.

[0057] Cells stained with CD22[5E8.1.8] accounted for 93.976% of the total cells ( image 3 ).

Embodiment 3

[0059] Referring to the experimental method of Example 1, CD3[SK7] was detected, and CD3[SK7] expressed Mouse IgG1, kappa.

[0060] CD3[SK7] cells accounted for 0.352% of the total cells ( Figure 4 ).

[0061] Judging from the above examples, CD22[5E8.1.8] cells had the highest positive rate of secreting antibodies, followed by CD22[10F4.4.1], and CD3[SK7] cells had no positive cells secreting antibodies.

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Abstract

The method for rapidly detecting the antibody secreted by the hybridoma cell comprises the following steps: displaying an Fc segment of the antibody in the hybridoma cell in cytoplasm, analyzing the Fc segment in the cytoplasm, and judging the antibody specificity and the antibody generation capability of the cell. According to the method provided by the invention, the positive rate proportion of antibody cells secreting antibodies in some intracellular expression antibodies and hybridoma cells in the total cell number can be visually detected. The antibody secretion capability of each monoclonal cell and the non-specific component on the surface of the hybridoma cell can be detected.

Description

technical field [0001] The invention belongs to the field of cell biology, in particular to a kit for rapidly detecting whether hybridoma cells secrete antibodies. Background technique [0002] An antibody is an immunoglobulin (Ig) that specifically binds an antigen, which can be hydrolyzed by proteolytic enzymes. For example, it is hydrolyzed into two F(ab) segments and one Fc segment by papain. The F(ab) segment of the antibody can bind to the antigen, while the Fc segment functions to bind to the Fc receptor and mediate opsonization or antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity ). Since the Fc segment can be directly combined with enzymes or fluorescent dyes to label antibodies, it is not only the site of antibody rivets on the fixed plate in ELISA experiments, but also the site of recognition and binding of secondary antibodies in immunoprecipitation, western blotting and immunohistochemistry. [0003] Hybridoma ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6854G01N33/56966
Inventor 张洋褚建达张娅
Owner ZHEJIANG ZHENGXI BIOMEDICAL CO LTD
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