Monoclonal Antibody Production

Biology |

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General Aim of MAB Labs

One type of antibody derived from a single cloned B cell.

 

Method of Monoclonal Antibody Production

Hybridoma Technology

Learning Objectives (ILOs)

  • Practice sterile cell culture techniques for monoclonal antibody production.

  • 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 by following the mab production steps.
  • Screen hybridoma colonies for specific antibody production using ELISA in monoclonal antibody production.
  • Freezing hybridomas.
  • Understand how are monoclonal antibodies made in the lab.

Theoretical Background / Context

  • The Monoclonal Antibody Production process requires several months.

The General Procedure Includes the Following Monoclonal Antibody Development 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 for monoclonal antibody production.
  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.
 

Principle of Work

The Monoclonal Antibody Manufacturing Process:

  • The first step is Immunization, where a mouse 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’ through hybridoma technology and monoclonal antibodies production.
  • This fusion is done with the help of polyethylene glycol (PEG). 
  • Hybridomas should be able to produce the desired antibody through monoclonal antibodies hybridoma technology. 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 in the monoclonal antibody production workflow.

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