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Method for efficiently isolating single antigen-specific b-lymphocytes from spleen cells

A technology for B lymphocytes and spleen cells, applied in the field of efficient separation of single antigen-specific B lymphocytes, can solve the problems of increasing the difficulty of B lymphocytes and the lack of types of antibody tools, ensuring monoclonality, solving instability, The effect of quick filtering

Active Publication Date: 2020-04-24
优睿赛思(武汉)生物科技有限公司
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  • Summary
  • Abstract
  • Description
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Problems solved by technology

[0007] 3. At present, there are few antibody tools targeting CD molecules on the surface of rabbit B lymphocytes, which further increases the difficulty of sorting B lymphocytes at specific stages

Method used

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  • Method for efficiently isolating single antigen-specific b-lymphocytes from spleen cells
  • Method for efficiently isolating single antigen-specific b-lymphocytes from spleen cells
  • Method for efficiently isolating single antigen-specific b-lymphocytes from spleen cells

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Embodiment 1

[0032] 1. Pre-labeling of antigenic substances:

[0033] 1.1 If the antigenic substance is a protein, use long-chain biotin purchased from Pierce in the United States to couple according to the standard procedure recommended by the manufacturer. After the coupling is completed, dialyze out the unlabeled biotin; if the antigenic substance is a polypeptide or a small molecule compound , the biotin molecule is pre-coupled at the time of synthesis. The antigen in this example is human C-reactive protein (CRP) (purchased from Fitzgerald Industries International)

[0034] 1.2 Conjugate the biotin-labeled antigen with a small molecule fluorescent dye. In this example, Cy5-coupled avidin is used to couple the antigenic substance; according to the size of the antigenic substance, calculate the required Cy5-coupled avidin The recommended amount is that the molar ratio of Cy5-coupled avidin and biotin-coupled antigen is greater than five to one, and the coupling reaction is carried out ...

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Abstract

The invention discloses a method for efficiently separating single antigen-specific B lymphocyte from spleen cells. High-throughput rapid screening of single antigen-specific B lymphocyte is realizedby negative screening adopting multiple lymphocyte surface marker antibodies, establishment of a rapid fluorescence marked antigen substance method and high-specificity screening by use of a fluorescence marked antigen substance, and the positive rate of the finally obtained antibody-specific B lymphocyte is increased from less than 5% in a traditional hybridoma method to 30%. Therefore, the scheme can shorten monoclonal antibody development time to a great extent and greatly increase the number and diversity of obtained monoclonal antibodies.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for efficiently isolating single antigen-specific B lymphocytes from spleen cells. Background technique [0002] B lymphocytes, referred to as B cells for short, are mainly derived from pluripotent stem cells in the bone marrow. The differentiation process of mammalian B cells can be divided into five stages: pre-B cells, immature B cells, mature B cells, activated B cells and plasma cells. Among them, the differentiation of pre-B cells and immature B cells is antigen-independent, and its differentiation process is mainly carried out in the bone marrow; mature B cells will enter the peripheral blood through the circulation of the lymphatic system, and can further differentiate after being stimulated by antigens Plasma cells that synthesize and secrete antibodies. Plasma cells can produce immunoglobulin IgG specific to an antigen and release it into body fluids, which becom...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0781
CPCC12N5/0635C12N2509/00
Inventor 娄阳吴海
Owner 优睿赛思(武汉)生物科技有限公司
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