Monoclonal Antibodies and its Production

Introduction

This tutorial provides a step-by-step guide on the production of monoclonal antibodies, a crucial tool in biotechnology and medicine. Understanding the process of creating these antibodies is essential for applications such as diagnostics, therapeutics, and research.

Step 1: Understanding Monoclonal vs. Polyclonal Antibodies

• Monoclonal Antibodies:

  • Produced by cloning a single type of white blood cell.
  • Have monovalent affinity, binding only to one specific epitope of an antigen.

• Polyclonal Antibodies:

  • Produced by multiple plasma cell lineages.
  • Bind to several different epitopes on the same antigen.

• Bispecific Monoclonal Antibodies:

  • Engineered to target two different epitopes, enhancing therapeutic potential.

Step 2: Identifying Antigen-Specific Cells

• Identify antigen-specific plasma or plasmablast cells (ASPC) that produce antibodies for the target antigen.
• Use techniques such as flow cytometry to isolate these cells based on their surface markers.

Step 3: Fusing Cells to Create Hybridomas

• Fusion Process:

  • Fuse identified ASPCs with myeloma cells to create hybridomas.
  • Myeloma cells are used because they can grow indefinitely and have lost the ability to synthesize HGPRT, an enzyme necessary for nucleic acid synthesis.

• Fusion Agent:

  • Use polyethylene glycol (PEG) to facilitate the fusion of adjacent plasma membranes.

Step 4: Selecting Hybridomas with HAT Medium

• HAT Medium:

  • Contains hypoxanthine, aminopterin, and thymidine, which creates a selective environment for hybridoma cells.

• Mechanism:

  • Myeloma cells cannot grow in this medium due to the lack of HGPRT.
  • Spleen cells cannot survive indefinitely due to their limited lifespan.
  • Only hybridomas, which combine the immortal characteristics of myeloma cells and the HGPRT-producing capabilities of spleen cells, can thrive.

Step 5: Screening and Cloning Hybridomas

• After establishing hybridoma cultures, screen for those producing the desired antibody.
• Clone the selected hybridomas to ensure a uniform population of antibody-producing cells.

Step 6: Antibody Production and Purification

• Once a hybridoma is selected, scale up the culture to produce large quantities of antibodies.
• Purify the antibodies using methods such as affinity chromatography, which isolates antibodies based on their binding properties.

Conclusion

The production of monoclonal antibodies involves a series of critical steps from cell identification to hybridoma creation and antibody purification. Mastering this process opens doors to significant advancements in medical and research applications. For further exploration, consider diving deeper into the specific techniques used for cell culture and antibody purification methods.