Method for preparing hybridoma by enriching mouse plasma cells with CD138<+> biomarker and application

A technology of biomarkers and plasma cells, which is applied in the field of plasma cells, can solve problems such as increasing the challenge of R&D projects, specific plasma cell interference, and working hours of R&D personnel, so as to improve work efficiency and work quality, increase the positive rate, The effect of reducing manpower

Active Publication Date: 2021-04-09
SHANGHAI WUXI BIOLOGIC TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And the screening in a short period of time leads to the working hours of the R&D personnel, and the work intensity is extremely high
Even some projects contain very little plasma cell components (less than 0.5%), and the fused specific plasma cells are severely disturbed by other mixed tissue cells, resulting in a large number of invalid hybridomas, which greatly increases the challenge of R&D projects and even leads to R&D project failed

Method used

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  • Method for preparing hybridoma by enriching mouse plasma cells with CD138&lt;+&gt; biomarker and application
  • Method for preparing hybridoma by enriching mouse plasma cells with CD138&lt;+&gt; biomarker and application
  • Method for preparing hybridoma by enriching mouse plasma cells with CD138&lt;+&gt; biomarker and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Enrichment of mouse plasma cells

[0045] There are only a handful of mouse plasma cell enrichment kits currently on the market, mainly including: STEMCELLEasySep.Mouse CD138 Positive Selection Kit (Catalog#18957) and Miltenyi BiotecCD138+Plasma Cell Isolation Kit (no.130-092-530), The former is used for the enrichment of plasma cells in the present invention.

[0046] Plasma cell enrichment process materials:

[0047] Reagent name Specification store Mouse CD138Positive Selection Cocktail 1×1mL 2-8℃ Dextran RapidSpheres 1×1mL 2-8℃ Mouse FcR Blocker 2×0.5mL 2-8℃

[0048] See: STEMCELL EasySep.Mouse CD138 Positive Selection Kit (Catalog#18957)

[0049] figure 2 and image 3 Spleen or spleen and lymph node tissues of different Balb / c mice after immunization were taken respectively, and the mixed immune tissue cells were separated by grinding with glass rods, and the operation was as follows:

[0050] 1. Prepare Re...

Embodiment 2

[0059] Example 2: Mouse plasma cell fusion and screening

[0060] Spleen tissue of immunized mice was taken, ground with a glass rod, and a part of the cells were treated with erythrocyte lysate for traditional hybridoma fusion with mouse myeloma cells (in traditional hybridoma fusion, mixed cells treated with erythrocyte lysate, Cell washing, counting, pronase buffer treatment and fusion steps in mouse myeloma cells can refer to the following steps). A part of the cells were enriched for plasma cells using the mouse Mouse CD138 positive kit (as described in the step in Example 1 above). The enriched plasma cells were then used for cell fusion with mouse myeloma cells.

[0061] Plasma cell fusion process materials:

[0062]

[0063] The cell fusion method is exemplified by electrofusion, and the specific steps are as follows:

[0064] 1. Enrichment of mouse plasma cells according to the steps of Example 1. Use DMEM basal medium to collect the enriched plasma cells on th...

Embodiment 3

[0079] Example 3: Comparison of mouse plasma cell fusion vs traditional hybridoma fusion-positive hybridoma antibody epitope binding diversity and subtype diversity

[0080] 1, as embodiment 2 item 2 (corresponding Figure 5 The antibodies secreted by the positive hybridomas screened in ) were tested for subtypes, and the results showed that the positive hybridoma antibodies screened by plasma cell fusion showed more subtype diversity than the hybridoma antibodies screened by traditional hybridomas. Specific antibodies are produced, and the antibodies produced are more diverse. The results are shown in Table 1.

[0081] 2, as embodiment 2 item 2 (corresponding Figure 5 The antibody secreted by the positive hybridoma screened in ) was tested with BMK for specific antigen and antibody binding epitope. The results showed that the positive hybridoma antibody screened by plasma cell fusion showed more Multiple antigen-binding epitope diversity. Antigen-binding epitopes are hig...

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Abstract

The invention discloses a method for preparing hybridoma by enriching mouse plasma cells with a CD138<+> biomarker. The method comprises the following steps of: S1, enrichment: enriching the mouse plasma cells with the plasma cell surface CD138<+> biomarker; and S2, fusion: collecting the enriched mouse plasma cells, and carrying out cell electrofusion on the mouse plasma cells and mouse myeloma cells. According to the invention, the mouse plasma cells are enriched by utilizing the CD138<+> biomarker on the surfaces of the plasma cells, so as to greatly improve the purity of the plasma cells, thereby obtaining a large number of hybridoma cells capable of secreting specific antibodies, and greatly improving the positive rate of the specific antibodies in the screening process. Meanwhile, the number of cells fused by initial cells and the mouse myeloma cells is greatly reduced, so that the number of bed boards is greatly reduced, and high-throughput screening and human and material costs are reduced.

Description

technical field [0001] The invention relates to the technical field of preparation of plasma cells and hybridoma cells-monoclonal antibodies, in particular to a method for preparing hybridomas by using CD138+ biomarkers to enrich mouse plasma cells and its application. Background technique [0002] Hybridoma-monoclonal antibody technology was invented by Kohler and Milstein in 1975 and won the Nobel Prize in Physiology and Medicine in 1984. The main process of traditional hybridoma technology is: 1) immunize mice with specific antigens; 2) take splenocytes from immunized mice and mouse myeloma cells for cell fusion, and the new cells formed after the fusion of the two cells in vitro are both With the ability of unlimited proliferation of myeloma cells and the ability of B cells to secrete antibodies, the fusion cells are called hybridoma cells (hybridoma cells); 3) High-throughput, quickly screen a large number of hybridomas to determine the positive for specific antigens h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/06C12N5/20C07K16/18A61K39/395A61P35/00
CPCC12N5/163C07K16/18A61P35/00A61K2039/505
Inventor 陆青玲王德宁罗芳芳施利波姚晓玲梅芹顾继杰
Owner SHANGHAI WUXI BIOLOGIC TECH CO LTD
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