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Screening method of monoclonal antibody cells for expressing, secreting and distinguishing natural sample

A technology of monoclonal antibodies and screening methods, applied in analytical materials, instruments, measuring devices, etc., can solve the problems of identifying natural samples, unable to screen and identify cells of natural samples in a targeted manner, and unable to detect antibodies, etc.

Inactive Publication Date: 2020-10-23
CUSABIO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of method can only detect whether the cell culture supernatant can recognize recombinant proteins or immunogenic proteins, and cannot detect which cells secrete antibodies that can recognize natural samples
Therefore, the cells that recognize natural samples cannot be screened in a targeted manner, but only after the titer is detected, some cells are randomly retained, and then the cells that recognize natural samples can be detected after the antibody is prepared and purified; most of them have potential The cells are likely to be discarded when randomly reserved at that time, which leads to the blindness and randomness of cell selection, thus increasing the difficulty and cost of project development

Method used

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  • Screening method of monoclonal antibody cells for expressing, secreting and distinguishing natural sample

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Experimental program
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Effect test

Embodiment 1

[0038] The screening method of the monoclonal antibody cells expressing, secreting and recognizing natural samples described in the present invention is as follows:

[0039] (1) Preparation of cell supernatant: select TRF protein for subcutaneous immunization of mice, and when the immunization reaches 5 immunizations, a small amount of blood is collected from the mice; the collected blood is centrifuged to obtain mouse serum; and then the mice are Serum was tested for titer by ELISA, and when the titer reached 1:10000, the mouse was selected for cell fusion operation. Spleen cells and lymphoma cells (SP2 / 0) of the mouse were collected, cell fusion was performed under the action of PEG1450, and the fused cells were plated and cultured in 96-well plates (about 50 plates). Under the action of HAT medium, those cells that can secrete antibodies and can be immortalized are retained, while the rest of the cells die in a large number about 7 days after culture. On the 5th day after ...

Embodiment 2

[0049] The screening method of the monoclonal antibody cells expressing, secreting and recognizing natural samples described in the present invention is as follows:

[0050] (1) Preparation of cell supernatant: TRF protein was selected for subcutaneous immunization of rabbits. When the immunization reached 5 times, a small amount of blood was collected from the rabbits; the collected blood was centrifuged to obtain rabbit serum; then ELISA was performed on the rabbit serum. When the titer reaches 1:10000, the rabbit is selected for cell fusion operation. Spleen cells and lymphoma cells (SP2 / 0) of the rabbit were collected, cell fusion was performed under the action of PEG1450, and the fused cells were plated and cultured in 96-well plates (about 50 plates). Under the action of HAT medium, those cells that can secrete antibodies and can be immortalized are retained, while the rest of the cells die in a large number about 7 days after culture. On the 5th day after culturing, th...

Embodiment 3

[0060] The screening method of the monoclonal antibody cells expressing, secreting and recognizing natural samples described in the present invention is as follows:

[0061] (1) Preparation of cell supernatant: select TRF protein for subcutaneous immunization of mice, and when the immunization reaches 5 immunizations, a small amount of blood is collected from the mice; the collected blood is centrifuged to obtain mouse serum; and then the mice are Serum was tested for titer by ELISA, and when the titer reached 1:10000, the mouse was selected for cell fusion operation. Spleen cells and lymphoma cells (SP2 / 0) of the mouse were collected, cell fusion was performed under the action of PEG1450, and the fused cells were plated and cultured in 96-well plates (about 50 plates). Under the action of HAT medium, those cells that can secrete antibodies and can be immortalized are retained, while the rest of the cells die in a large number about 7 days after culture. On the 5th day after ...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a screening method of monoclonal antibody cells for expressing, secreting and distinguishing a natural sample. Thescreening method comprises the following steps of: preparing an enzyme-labeled microporous plate coated with a secondary antibody; carrying out sealing treatment on the enzyme-labeled microporous plate coated with the secondary antibody to obtain a sealed enzyme-labeled microporous plate coated with the secondary antibody; sequentially adding a cell supernatant, a natural sample, an enzyme-labeledantibody corresponding to the natural sample and a substrate developing reagent into the sealed enzyme-labeled microporous plate coated with the secondary antibody to obtain a to-be-detected enzyme-labeled microporous plate; and carrying out absorbance detection on the to-be-detected enzyme-labeled microporous plate to obtain a screening result, wherein the concentration of the enzyme-labeled antibody corresponding to the natural sample is 0.5-5 [mu]g / mL. According to the screening method disclosed by the invention, monoclonal antibody cells secreting and distinguishing natural samples can berapidly detected in a cell culture stage, so that cell screening, accepting and rejecting in the process are not random any more, and which cells can be reserved can be judged in a cell supernatant screening stage.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a screening method for cells expressing and secreting monoclonal antibodies that recognize natural samples. Background technique [0002] At present, in the research and development of the preparation of monoclonal antibodies, the method of cell fusion is usually adopted. In the process of using the cell fusion method, due to the large number of fused cells, a lot of screening work needs to be done in the cell culture screening stage in the later stage. In this type of screening work, most of the screening is oriented towards the identification of recombinant proteins, and the cell culture supernatants at this stage are all tested by ELISA. The general steps are as follows: It was placed on the enzyme-labeled plate; blocked with excess protein; added to the cell culture supernatant; added to the corresponding enzyme-labeled secondary antibody and added to the substrate f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/535G01N33/543
CPCG01N33/535G01N33/54306G01N33/56966G01N33/577
Inventor 余焜舒芹赵愿安
Owner CUSABIO TECH LLC