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
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
Mansi Popat & Japan Raval
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