Hybridoma Technology and monoclonal antibodies

Hybridoma technology is a method used to produce large quantities of identical
antibodies (monoclonal antibodies).
It involves fusing a specific type of immune cell (B-cell) with a cancerous immune cell
(myeloma cell), creating a hybrid cell that can both produce antibodies and
proliferate indefinitely. These hybrid cells are called hybridomas.

1. Hybridoma Technology and monoclonal antibodies Dr. Vividha raunekar

2. Hybridoma technology is a method used to produce large quantities of identical antibodies (monoclonal antibodies). It involves fusing a specific type of immune cell (B-cell) with a cancerous immune cell (myeloma cell), creating a hybrid cell that can both produce antibodies and proliferate indefinitely. These hybrid cells are called hybridomas . Here’s a step-by-step outline of the process: 1. Immunization Phase In this initial step, an animal (usually a mouse, but sometimes rabbits or other animals) is repeatedly injected with the antigen , the molecule you wish to generate antibodies against. This antigen could be anything like a protein, peptide, or even a small molecule that acts as an immunogen . The mouse’s immune system recognizes the antigen as foreign, and its B-cells (a type of white blood cell) start producing antibodies to specifically bind to this antigen. Booster injections are often administered to ensure a robust immune response. This can improve the quality and specificity of the antibodies produced. Key Concepts: Polyclonal Response : The animal will produce many different types of antibodies (polyclonal), each recognizing different parts (epitopes) of the antigen. Humoral Immunity : This refers to the component of the immune response mediated by B-cells that produce antibodies.

3. 2. Isolation of B-cells After the immune system generates sufficient antibodies, the animal is euthanized, and its spleen is removed. The spleen is a critical organ for the immune system and is rich in B-cells. These spleen cells contain the desired antigen-specific B-cells that produce antibodies targeting the antigen introduced earlier. The B-cells extracted from the spleen are naturally short-lived and will die after a few days unless they are fused with cancer cells (myeloma cells) to become immortal. 3. Fusion with Myeloma Cells This step involves combining the antigen-specific B-cells with myeloma cells , a type of immortalized cancer cell derived from plasma cells. Myeloma cells can divide indefinitely, a crucial characteristic because B-cells cannot. Fusion Process: Polyethylene Glycol (PEG) or Sendai Virus is typically used to promote fusion between the cell membranes of B-cells and myeloma cells. PEG induces temporary fusion by dehydrating the cells, allowing the membranes to merge. The result is a hybridoma : a hybrid cell that combines the ability of the B-cell to produce antibodies and the ability of the myeloma cell to grow indefinitely.

4. 4. Selection in HAT Medium The next step is selection . Not all fusions between B-cells and myeloma cells are successful, and some cells will not fuse properly. To isolate the successfully fused hybridoma cells, the cells are grown in a special culture medium called HAT medium . HAT Medium Components : Hypoxanthine : A precursor for DNA synthesis. Aminopterin : Blocks the de novo synthesis pathway of nucleotides. Thymidine : A nucleoside necessary for DNA replication. In this medium: Unfused myeloma cells die because they lack the enzyme HGPRT (hypoxanthine-guanine phosphoribosyltransferase ) and cannot survive on the salvage pathway alone (blocked by aminopterin ). Unfused B-cells die naturally since they are not immortal. Successfully fused hybridomas survive because they can use the salvage pathway from the B-cell. 5. Screening for Antibody Production Once the hybridomas have been selected, the next step is to screen them for the production of the desired antibody . The goal here is to identify the hybridoma that produces an antibody specific to the antigen of interest.

5. 6. Cloning of Hybridomas Once the desired antibody-producing hybridoma is identified, it needs to be cloned to ensure that all the cells are identical and produce the same monoclonal antibody. Limiting dilution cloning is a common technique. Hybridoma cells are diluted in such a way that each well of a 96-well plate contains only one cell. This single cell grows into a colony of identical cells, all producing the same antibody. Re-cloning may be done to ensure purity and uniformity. 7. Large-scale Production of Monoclonal Antibodies The selected hybridoma can now be used to produce large amounts of monoclonal antibodies. These can be harvested either from: In vitro culture : The hybridomas are grown in bioreactors or flasks, and the monoclonal antibodies are harvested from the culture supernatant. In vivo (in animals): Hybridoma cells are injected into the peritoneal cavity of mice, where they form tumors and produce large amounts of antibodies in the mouse’s ascitic fluid. The antibodies are then purified from this fluid. Enzyme-linked Immunosorbent Assay (ELISA) is a commonly used technique. It works by coating a plate with the antigen, then adding the hybridoma supernatant (the liquid in which hybridomas grow) to see which hybridoma produces an antibody that binds to the antigen. The bound antibody is detected by using an enzyme-linked secondary antibody, which produces a colorimetric change, indicating a positive result. Flow cytometry or radioimmunoassay may also be used for screening.

6. Applications of Hybridoma Technology 1. Diagnostic ELISA Tests : Monoclonal antibodies are used in enzyme-linked immunosorbent assays to detect the presence of specific proteins, hormones (e.g., hCG in pregnancy tests), or pathogens (e.g., HIV, COVID-19 detection kits). Flow Cytometry : For identifying specific cell populations in blood, using fluorescently labeled monoclonal antibodies that bind to cell surface markers. 2. Therapeutic Cancer Therapy : Monoclonal antibodies can target specific antigens on cancer cells. For example, Herceptin ( trastuzumab ) is used to treat HER2-positive breast cancer, while Rituximab targets CD20 in B-cell lymphomas. Autoimmune Diseases : Monoclonal antibodies like infliximab (anti-TNF- α) are used to treat rheumatoid arthritis, Crohn’s disease, and psoriasis. Infectious Diseases : Monoclonal antibodies are developed to neutralize pathogens, such as RSV (Respiratory Syncytial Virus) or COVID-19. 3. Research Western Blotting : Monoclonal antibodies are used to detect specific proteins after separation by gel electrophoresis. Immunohistochemistry (IHC) : Monoclonal antibodies are used to detect specific antigens in tissue samples. Flow Cytometry : Monoclonal antibodies are labeled with fluorescent dyes to detect specific proteins on the surface of cells.

7. Humanization of Monoclonal Antibodies Mouse-derived monoclonal antibodies are often recognized by the human immune system as foreign and can cause immune reactions. To overcome this issue, humanized monoclonal antibodies are created by replacing most of the mouse antibody sequence with human sequences, leaving only the antigen-binding regions intact. Chimeric Antibodies : Partially humanized antibodies where the constant region is human but the variable region remains mouse-derived. Fully Human Antibodies : Produced using recombinant DNA technology or by using genetically modified mice with human immunoglobulin genes. Challenges and Limitations Cost : Hybridoma technology can be expensive, especially when scaling up for large-scale production. Time : It takes several months to produce and screen hybridomas for specific monoclonal antibodies. Ethical Concerns : The use of animals, particularly for the production of monoclonal antibodies, raises ethical concerns.

8. Monoclonal and Polyclonal Antibodies 1. Monoclonal Antibodies ( mAbs ) Definition: Monoclonal antibodies are homogeneous antibodies produced by a single type of immune cell (B-cell clone), which means they are specific to one unique epitope (the precise part of the antigen they bind to). Production Process ( Hybridoma Technology): Monoclonal antibodies are typically produced using hybridoma technology . The key steps include: Immunization : An animal (commonly a mouse) is injected with the antigen. B-cell Harvesting : Antibody-producing B-cells are isolated from the spleen. Fusion with Myeloma Cells : B-cells are fused with immortal myeloma cells to form hybridomas . Selection : Hybridomas are grown in HAT medium, and only successfully fused hybridomas survive. Screening : Hybridomas that produce the desired antibody are selected. Cloning and Expansion : The selected hybridomas are cloned to ensure uniform antibody production. Both monoclonal and polyclonal antibodies are essential tools in immunology, diagnostics, and therapeutic fields. They differ in their production methods, characteristics, and applications. Here's an in-depth comparison:

9. Characteristics: Specificity : Monoclonal antibodies bind to a single, specific epitope of an antigen. Homogeneity : All antibodies produced are identical, resulting in consistent and reproducible performance. Purity : Monoclonal antibodies are generally of high purity, as they come from a single B-cell clone. Advantages: High specificity : Monoclonal antibodies target only one specific epitope, minimizing cross-reactivity. Reproducibility : Because they are derived from a single clone, they can be consistently reproduced over time. Therapeutic Use : Monoclonal antibodies can be tailored for specific therapeutic targets, such as cancer cells or autoimmune disease markers.

10. Disadvantages: Time-consuming production : It can take months to generate a monoclonal antibody. Cost : The production process is expensive. Specificity limitation : Monoclonal antibodies recognize only one epitope, which can be a disadvantage if the antigen mutates or is highly variable. Immunogenicity : Antibodies derived from animals may cause immune responses when used in humans (though humanized or fully human monoclonal antibodies can mitigate this). Applications: Therapeutics : Used in cancer treatment (e.g., Herceptin, Rituximab), autoimmune diseases (e.g., Adalimumab for rheumatoid arthritis), and infectious diseases (e.g., Palivizumab for RSV). Diagnostics : Used in highly specific diagnostic tests like ELISA , Western Blot , and Flow Cytometry to detect proteins, pathogens, or toxins. Research : Used in laboratory techniques for identifying and quantifying specific proteins, peptides, or molecules.

11. 2. Polyclonal Antibodies ( pAbs ) Definition: Polyclonal antibodies are a mixture of antibodies produced by different B-cell clones in response to an antigen. These antibodies can recognize and bind to multiple epitopes on the same antigen. Production Process: Polyclonal antibodies are generated in living animals (like rabbits, goats, or mice): Immunization : The animal is injected with the antigen. B-cell Activation : Multiple B-cell clones in the animal’s immune system recognize different epitopes on the antigen and produce antibodies. Blood Collection : After the immune response has developed, blood is collected from the animal. Antibody Isolation : The antibodies are purified from the blood serum. Characteristics: Multiple Epitope Recognition : Polyclonal antibodies recognize several different epitopes on the antigen. Heterogeneity : Since they come from many B-cell clones, they are a mixture of different antibodies with varying specificities and affinities.

12. Advantages: Broad reactivity : Polyclonal antibodies recognize multiple epitopes, making them effective even if the antigen has slight variations or mutations. More robust binding : The recognition of multiple epitopes can result in stronger binding to the target antigen. Faster production : Polyclonal antibodies can be generated relatively quickly (a few weeks) and are more cost-effective than monoclonal antibodies. Versatility : They are effective in recognizing proteins that might have undergone post-translational modifications or are in native forms. Disadvantages: Batch variability : Because polyclonal antibodies are produced in live animals, each batch may differ slightly, making reproducibility a challenge. Lower specificity : Polyclonal antibodies may cross-react with other antigens, leading to potential non-specific binding in some applications. Limited supply : The production of polyclonal antibodies is finite; once an animal's antibody production declines, new animals must be used.

13. Applications: Diagnostics : Widely used in assays where high sensitivity is important. Polyclonal antibodies can detect a variety of antigen forms and mutations. Immunohistochemistry (IHC) : Used to stain and visualize proteins in tissue samples. Immunoprecipitation : Used to capture proteins from complex mixtures because of their ability to bind multiple epitopes. Western Blotting : Due to their broad epitope recognition, polyclonal antibodies are effective in detecting denatured or fragmented proteins.

14. Feature Monoclonal Antibodies (mAbs) Polyclonal Antibodies (pAbs) Source Produced by a single B-cell clone (hybridoma) Produced by multiple B-cell clones in an animal Epitope Recognition Recognizes a single epitope Recognizes multiple epitopes on the same antigen Specificity Highly specific, binds to one site on the antigen Less specific, binds to multiple sites on the antigen Homogeneity Homogeneous (all antibodies are identical) Heterogeneous (mix of different antibodies) Production Method Time-consuming and expensive (Hybridoma technology) Quick and cost-effective (animal immunization and serum collection) Batch Consistency High consistency across batches Variability between batches Reactivity May not work well with antigen variations Works well with antigen variants and mutations Applications Therapeutics, diagnostics, and research (when high specificity is needed) Diagnostics, research, and techniques requiring broader detection Immunogenicity in Humans Can be immunogenic if not humanized Less suitable for therapeutic use in humans Key Differences Between Monoclonal and Polyclonal Antibodies

15. Summary Monoclonal antibodies are highly specific and bind to a single epitope, making them ideal for applications that require precision, such as therapeutic treatments for specific diseases or targeted diagnostic tests. Their production is complex but results in highly consistent and reproducible antibodies. Polyclonal antibodies are more versatile, recognizing multiple epitopes on an antigen, which makes them useful in diagnostic and research applications that need robust and broad detection. They are faster and cheaper to produce but come with the challenge of batch variability and lower specificity.

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