Hybridoma Technology: Lab made Antibodies!

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Introduction

  • Hybridoma technology is a method for producing large quantities of monoclonal antibodies (MAbs) by fusing a specific type of white blood cell (B-cell) with a myeloma (cancer) cell.
  • This technology allows for the production of antibodies that are identical (monoclonal), providing high specificity and consistency in binding to the target antigen.

Historical Background

  • The technique was first developed by Georges Köhler and César Milstein in 1975, for which they were awarded the Nobel Prize in 1984.
  • Hybridoma technology revolutionized immunology, leading to significant advances in diagnostic and therapeutic applications.

Procedure


Fig 1: General Procdure

 

  1. Immunization of Host Animal:
    • Objective: The goal is to induce a robust immune response in the host animal (commonly a mouse), leading to the production of B-cells that generate antibodies specific to the antigen of interest.
    • Process:
      • The antigen, typically a protein or a peptide, is injected into the mouse, often in combination with an adjuvant to enhance the immune response.
      • The injections are administered at intervals (boosters) to ensure a strong and sustained antibody production.
      • After a series of immunizations, blood samples are taken from the mouse to test for the presence of the desired antibodies.
  2. Isolation of B-Cells from Spleen:
    • Objective: Harvest B-cells from the spleen of the immunized mouse, as these cells are responsible for producing the antibodies.
    • Process:
      • The mouse is euthanized, and the spleen is aseptically removed.
      • The spleen is mechanically dissociated, often using a syringe or a tissue grinder, to release the B-cells into a suspension.
      • The cell suspension is filtered to remove debris and then centrifuged to concentrate the B-cells.
  3. Fusion with Myeloma Cells:
    • Objective: To create hybrid cells (hybridomas) that combine the antibody-producing capability of B-cells with the immortality of myeloma cells.
    • Materials:
      • Myeloma Cells: These are cancerous plasma cells that have lost the ability to produce antibodies but can proliferate indefinitely.
      • Fusion Agent: Polyethylene glycol (PEG) is commonly used to induce the fusion of B-cells and myeloma cells by promoting the merging of their membranes.
    • Process:
      • The B-cells and myeloma cells are mixed in a defined ratio in the presence of PEG.
      • PEG facilitates the fusion of cell membranes, leading to the formation of hybridoma cells, which inherit the desirable traits from both parent cells.
      • The fusion mixture is carefully washed to remove excess PEG, as it can be toxic to cells.
  4. Selection of Hybridoma Cells:

 


Fig 2: Production of hybridomas

    • Objective: To selectively grow only the successfully fused hybridoma cells while eliminating unfused cells.
    • Selection Medium:
      • HAT Medium: A special culture medium containing Hypoxanthine, Aminopterin, and Thymidine (HAT) is used. Myeloma cells used for fusion are typically deficient in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT).
      • In the HAT medium, only cells that have successfully fused (i.e., hybridomas) can survive because they inherit the HGPRT enzyme from the B-cells, allowing them to bypass the blockage caused by aminopterin in the purine synthesis pathway.
    • Process:
      • After fusion, cells are placed in HAT medium.
      • Unfused B-cells die naturally due to their limited lifespan, while unfused myeloma cells perish because they cannot survive in HAT medium.
      • Only hybridoma cells can grow and multiply under these conditions.
  1. Screening and Identification of Desired Hybridomas:
    • Objective: To identify hybridomas that produce the monoclonal antibody specific to the antigen of interest.
    • Screening Methods:
      • ELISA (Enzyme-Linked Immunosorbent Assay): A common technique where the supernatant from hybridoma cultures is tested for the presence of antibodies that bind specifically to the antigen.
      • Flow Cytometry: Used in some cases to detect and quantify antibody production by individual hybridoma cells.
      • Western Blot: May be used to confirm the specificity and binding properties of the antibodies produced.
    • Process:
      • Hybridoma cells are grown in multi-well plates, and the supernatant from each well is tested.
      • Wells containing cells that produce the desired antibody are identified, and these hybridomas are selected for further development.
  2. Cloning of Hybridomas:
    • Objective: To obtain a pure population of cells producing a single, uniform monoclonal antibody.
    • Cloning Techniques:
      • Limiting Dilution Cloning: The selected hybridomas are diluted and plated such that only one cell is deposited per well, ensuring that each well contains a monoclonal population derived from a single hybridoma.
      • Soft Agar Cloning: Alternatively, cells can be embedded in a soft agar medium to facilitate the growth of colonies derived from individual hybridomas.
    • Process:
      • The cloned hybridomas are expanded in culture, and the supernatant is again tested to confirm the production of the desired monoclonal antibody.
  3. Expansion and Production of Monoclonal Antibodies:
    • Objective: To produce large quantities of the desired monoclonal antibody.
    • Process:
      • Hybridomas that produce the correct antibody are expanded in larger culture flasks or bioreactors.
      • The culture conditions are optimized to maximize antibody yield.
      • Antibodies are harvested from the culture medium, usually through filtration or centrifugation, followed by purification using techniques like protein A/G affinity chromatography.
  4. Characterization and Quality Control:
    • Objective: To ensure that the produced monoclonal antibodies meet the required standards for specificity, purity, and activity.
    • Characterization Techniques:
      • Affinity Measurement: Techniques like Surface Plasmon Resonance (SPR) or ELISA can be used to measure the binding affinity of the antibodies.
      • Specificity Testing: Cross-reactivity with other antigens is tested to ensure the monoclonal antibody is specific.
      • Purity Assessment: SDS-PAGE and Western blotting are often employed to assess the purity of the antibodies.
    • Process:
      • Detailed testing is performed on the final antibody product, and any necessary modifications or additional purification steps are undertaken to meet the desired specifications.

Applications

  • Therapeutic Uses:
    • Monoclonal antibodies are used in the treatment of various diseases, including cancer, autoimmune disorders, and infectious diseases.
  • Diagnostic Uses:
    • MAbs are widely used in diagnostic assays, such as ELISA and radioimmunoassay, due to their specificity.
  • Research:
    • In basic and applied research, MAbs serve as essential tools for identifying and quantifying biomolecules.

Advantages

  • High specificity and uniformity of monoclonal antibodies.
  • Ability to produce large quantities of antibodies consistently.
  • MAbs can be engineered to enhance their properties, such as reducing immunogenicity or increasing half-life.

Disadvantages

  • The initial process of hybridoma production is time-consuming and labor-intensive.
  • Hybridoma cells may lose the ability to produce antibodies over time.
  • The use of animals in the immunization process raises ethical concerns.

Future Prospects

  • Advances in genetic engineering and recombinant DNA technology are enhancing the production and functionality of monoclonal antibodies.
  • Humanization and chimeric antibodies are being developed to reduce immunogenicity when used in human therapy.
  • Hybridoma technology continues to evolve with ongoing research focused on improving efficiency and broadening its applications.


 Behind "The Biotechnology Journal"

Mansi Popat & Japan Raval


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