Production of Monoclonal Antibodies

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Production of Monoclonal Antibodies (mAB) Virtual Lab

General Aim of Production of Monoclonal Antibodies (mAB)

The production of one type of antibody derived from a single cloned B cell.

Production of Monoclonal Antibodies (mAB) Method

Hybridoma Technology.

Learning Objectives of Production of Monoclonal Antibodies (mAB)

  • Practice sterile cell culture techniques.

  • Evaluate the need to passage cells i.e. assess confluency.

  • Handle mice for immunization, including IV tail injection and intraperitoneal injection.

  • Practice serial dilution of serum.

  • Prepare single cell suspension out of the spleen of mice.

  • Perform the fusion technique using PEG.

  • Treat Hybridomas with HAT.

  • Prepare hybridomas for flow activated cell sorting FACS.

  • Screen hybridoma colonies for specific antibody production using ELISA.

  • Freezing hybridomas

Theory of Monoclonal Antibody Production Process

The production of a monoclonal antibody requires several months. The general procedure includes the following steps:
1. Immunization of mice with the antigen of interest.
2. Screening of mice antibody titer using ELISA. This step can tell if animals' immune systems responded to the antigens injected. It also indicates which  animal has the highest response.
3. Thawing and preparation of myeloma cells
4. Preparation of single cell suspension from mice spleens.
5. Fusion of antibody-producing B cells from spleens and myeloma cells.
6. Selective growth of the hybridoma in HAT.
7. Screening and sorting the hybridomas for producing the desired monoclonal antibody using FACS
8.Cloning of selected hybridomas.
9. Screening of hybridomas for antibody production using ELISA.
10.Freezing of the hybridoma.

Production of Monoclonal Antibodies (mAB) Principle of Work

In production of monoclonal antibodies (mAB) experiment, The first step is Immunization, where a mice is injected with the antigen, to which an antibody is to be generated. The rat’s immune system responds to the antigen by producing B cells. Each B cell produces a single type of antibody. B cells are isolated from the mouse’s spleen and fused with immortal myeloma cells producing ‘hybridoma cells’. This fusion is done with the help of polyethylene glycol (PEG). hybridomas should be able to produce the desired antibody. They are sorted using flow cytometry (FACS). Thus; hybridomas can be cloned into a 96 well plate in a 1cell/well ratio. When colonies become confluent, they can be screened for antibody production using ELISA.

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