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Separation method for plasma cells capable of secreting antigen specific antibodies

A separation method and plasma cell technology, applied in the field of monoclonal antibodies, can solve the problems of a large number of screening plates, heavy workload, and poor specificity, and achieve the effects of efficient preparation, high success rate, and high specificity

Inactive Publication Date: 2016-09-21
NINGBO ACCUTECH BIOSCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Then all the plasma cells are separated, and then gene cloning is carried out. Since the plasma cells of the antigen-specific antibody account for a relatively low proportion of the total plasma cells, the specificity is relatively poor and the efficiency is relatively low.
This technology is time-consuming, requires a lot of work, and requires a large number of screening plates. It is difficult to complete such screening manually, so a fully automatic machine is required to complete the screening.

Method used

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  • Separation method for plasma cells capable of secreting antigen specific antibodies

Examples

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Effect test

Embodiment 1

[0030] Example 1 Preparation of plasma cells

[0031] Mix Freund's complete adjuvant and human albumin solution at a ratio of 1:1 to make an emulsion. Immunize female Balb / c mice aged 6-8 weeks by intraperitoneal injection (100 microliters per mouse, containing 10 micrograms of human albumin). Two weeks later, mice were boosted with 10 micrograms of human albumin intraperitoneally injected with incomplete Freund's adjuvant. Two weeks later, blood was taken to determine the titer of antibodies. Mice were boosted by injecting 10 micrograms of human albumin through the tail vein. Four days later, the mice were sacrificed and spleen cells were isolated. Plasma cells were isolated from spleen cells using a plasma cell isolation kit (CD138+Plasma Cell Isolation Kit, mouse, Miltenyl Biotec).

Embodiment 2

[0032] Example 2 Preparation of bifunctional antibody

[0033] The bifunctional antibody was prepared by covalently cross-linking CD138 monoclonal antibody and mouse anti-IgG monoclonal antibody by chemical cross-linking method. BS3 (Thermo Scientific, Cat. #21580) was used as the cross-linking agent, and related experiments were carried out according to the operating instructions of the reagent. The cross-linked antibodies were separated using an affinity column.

Embodiment 3

[0034] Example 3 Crosslinking of Antigen and Magnetic Beads

[0035] The human albumin is cross-linked by chemical cross-linking method and magnetic beads. Preparation of antigen-magnetic bead conjugates for the isolation of plasma cells secreting antibodies specific to human albumin. Human albumin was dissolved in reaction buffer (0.05 M MES, 0.5 M NaCl, pH 6.0) to a final concentration of 1 mg / ml. Activate the carboxyl groups on the surface of the magnetic beads with a final concentration of 2 mM EDC and 5 mM sulfo-NHS. Mix the activated magnetic beads with the antigen, and incubate with shaking for 2 hours at room temperature. The magnetic beads were precipitated under a magnetic field, the supernatant was discarded, and the magnetic beads were washed twice with buffer.

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Abstract

The invention discloses a separation method for plasma cells capable of secreting antigen specific antibodies. The plasma cells capable of secreting the antigen specific antibodies are obtained through separating a plasma cell marker CD38 or CD138 mono-antibody and anti-IgG antibody cross-linked double-functional antibody and a magnetic bead which is crossly linked with a specific antigen. By adopting the separation method, the plasma cells capable of secreting the antigen specific antibodies can be accurately obtained, the working amount is extremely reduced, and the success rate is higher.

Description

technical field [0001] The invention belongs to the technical field of monoclonal antibodies, and in particular relates to a method for separating plasma cells secreting antigen-specific antibodies and a method for high-throughput preparation of antigen-specific monoclonal antibodies. Background technique [0002] Since the first antibody was applied to treatment in 1986, more than 50 antibody drugs have been used in clinical treatment. By 2020, about 70 antibody drugs will enter clinical application, and its market is expected to reach 125 billion US dollars. Plus detection antibodies for clinical diagnosis and antibodies for laboratory research. The market for antibodies will be well around $125 billion. Since most monoclonal antibodies are derived from mice, mouse antibodies are foreign proteins to humans and have high immunogenicity, which can easily cause the immune response of human anti-mouse antibodies, which greatly hinders the immune response of mice. The applica...

Claims

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Application Information

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IPC IPC(8): C12N5/0781C12N15/70C07K16/18C07K16/00C40B50/06C40B40/10C12R1/19
CPCC07K16/00C07K16/18C12N5/0635C12N2501/599C12N2509/10C40B40/10C40B50/06
Inventor 赖增祖叶欣徐健杨奎东
Owner NINGBO ACCUTECH BIOSCI LTD
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