SPA-mediated double antibody captured hybridoma technique

A double antibody, hybridoma technology, applied in the field of biomedicine, can solve the problems of affecting the fusion rate, cell survival rate, and high toxicity.

Pending Publication Date: 2018-07-31
翁炳焕
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, lymphocyte hybridoma technology still follows the myeloma cell and immune animal spleen cell hybridoma technology (PEG method) created by Kohler and Milstein (1975), which is based on cell fusion , the cell fusion agent used is polyethylene glycol (PEG1000~2000), although PEG is still the preferred fusion agent at present, but the toxicity of PEG to cells is very large, the higher the concentration, the greater the toxicity, and the effective fusion dose (50 %~80%) range of PEG is still very toxic to cells, which greatly affects the effective cell fusion rate and cell survival rate after fusion

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  • SPA-mediated double antibody captured hybridoma technique
  • SPA-mediated double antibody captured hybridoma technique
  • SPA-mediated double antibody captured hybridoma technique

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Experimental program
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Embodiment Construction

[0012] figure 1 It is a schematic diagram of SPA-mediated double-antibody capture aiding of the present invention.

[0013] figure 2 is the cell fusion diagram of the present invention.

[0014] exist figure 1 Among them, 1 represents the Staphylococcus aureus protein A (SPA) with IgGFc receptor, 2 represents the monoclonal antibody of the cells to be fused with numbers 6 and 7, and 3 represents the monoclonal antibodies of the cells to be fused with numbers 4 and 5 After the monoclonal antibody [2] is combined with the cells to be fused numbered 6 and 7, and the monoclonal antibody [3] is combined with the cells to be fused numbered 4 and 5, then through the connection of SPA, the numbered 4, The cells of 5, 6 and 7 are all favorable for fusion under the action of PEG.

[0015] exist figure 2 In the process, under the help of monoclonal antibody and / or SPA, the cells were screened by HAT for 2 weeks and photographed under an inverted microscope (40X) to obtain hybridom...

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Abstract

Provided is a SPA-mediated double antibody captured hybridoma technique applied to the field of biomedicine. The technique is characterized in that bone marrow cells (Ag1) and anti-myeloma cell antibodies (Ab1), and B cells (Ag2) and anti-B cell antibodies (Ab2) are respectively mixed at the ratio of 1:1, respectively undergo immune binding, are respectively formed into Ag1-Ab1 and Ag2-Ab2 conjugates; the two conjugates are mixed at the ratio of 1:1, half of IgGFc segment in the Ab1 of the Ag1-Ab1 conjugate is combined with a Fc receptor in the Ag2 of the Ag2-Ab2 conjugate, and a Ag1-Ab1-Ag2-Ab2 conjugate is obtained; half of the Ag1 is bonded to the Ag2 by the Ab1, staphylococcus aureus A protein (SPA) is added, an IgGFc receptor in the SPA is utilized to respectively bond non-combined IgGFc in the Ag1-Ab1 with non-combined IgGFc in the Ag2-Ab2, thereby under the effect of SPA, Ab1and Ab2, the distance of the myeloma cells and the B cells are closed specifically and proportionally, under the further effect of concanavalin A, IFN-gamma and PEG with low concentration, cell membranes are easily contacted and fused.

Description

technical field [0001] The invention relates to a SPA-mediated double-antibody capture hybridoma technology in the field of biomedicine. Background technique [0002] Hybridoma technology is lymphocyte hybridoma technology, also known as monoclonal antibody technology. [0003] Kohler and Milstein (1975) proved that myeloma cells were hybridized with spleen cells of immunized animals to form monoclonal antibodies that can secrete homogeneous and highly specific antibodies against the antigen. The technology is commonly known as hybridoma technology. Myeloma cells can be continuously passaged in vitro, while spleen cells are terminal cells that cannot be propagated in vitro. For example, if the myeloma cells of mice are hybridized with lymphocytes secreting certain antibodies or factors, the hybrid cells not only have the characteristics of unlimited proliferation of tumor cells, but also have the ability of lymphocytes to secrete specific antibodies or factors. Immune lym...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C12N15/02
CPCC12N5/163C12N15/02
Inventor 翁炳焕李兰娟温馨应俊毛愉婵
Owner 翁炳焕
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