Introduction
- Monoclonal antibodies (MAbs) are highly specific antibodies produced from a single clone of plasma cells.
- They recognize and bind to only one specific antigen or epitope.
- Monoclonal antibodies are widely used in diagnosis, treatment, research, and laboratory investigations.
- The discovery of monoclonal antibody technology revolutionized the field of immunology and biotechnology.
- Hybridoma technology made it possible to produce large quantities of identical antibodies.
- Hybridoma technology was developed by Georges Köhler and César Milstein in 1975.
- Monoclonal antibodies are highly pure, specific, and consistent in action.
- They are commonly used in cancer therapy, autoimmune diseases, and infectious disease diagnosis.
- Monoclonal antibodies are also used in pregnancy tests, blood grouping, and organ transplantation.
- Modern advances have led to the development of humanized and fully human monoclonal antibodies with fewer side effects.
What are Antibodies?
Antibodies are proteins produced by B lymphocytes or plasma cells in response to foreign substances called antigens. These antibodies bind specifically to antigens and help in their destruction.
Antibodies are also known as immunoglobulins (Ig).
There are two major types of antibodies:
1. Polyclonal Antibodies
These are produced by different clones of B cells and recognize multiple epitopes on the same antigen.
Characteristics
- Heterogeneous mixture
- Less specific
- Produced naturally during infection
- Shorter production consistency
2. Monoclonal Antibodies
These are produced from a single clone of B cells and recognize only one epitope of an antigen.
Characteristics
- Highly specific
- Homogeneous antibodies
- Produced artificially in laboratories
- Large-scale production possible
- Consistent quality
Monoclonal Antibodies
Monoclonal antibodies are identical immunoglobulins produced by a single clone of plasma cells against a specific antigenic determinant.
Important Features of Monoclonal Antibodies
- Recognize only one epitope
- Highly pure and specific
- Unlimited production possible
- Useful in diagnosis and therapy
- Less background reaction in laboratory tests
Hybridoma Technology
Hybridoma technology is the most important method for producing monoclonal antibodies.
It involves fusion of:
- Antibody-producing B lymphocytes
with - Immortal myeloma cells
The fused cells are called hybridoma cells.
These hybridoma cells possess:
- Ability to produce antibodies from B cells
- Unlimited growth property from myeloma cells
Thus, they continuously produce identical monoclonal antibodies.
Principle of Hybridoma Technology
The technique is based on combining the desirable properties of two different cells:
| Cell Type | Property |
|---|---|
| Plasma cell/B lymphocyte | Produces specific antibody |
| Myeloma cell | Immortal growth |
Fusion of these cells creates hybridoma cells capable of producing large quantities of specific monoclonal antibodies continuously.
Steps of Hybridoma Technology
1. Immunization of Mouse
A mouse is injected repeatedly with the desired antigen.
Purpose
- To stimulate immune response
- To produce antibody-forming B lymphocytes
Booster doses are given to increase antibody production.
2. Isolation of Spleen Cells
After adequate immunization:
- Spleen is removed from the mouse
- Spleen contains activated B lymphocytes producing antibodies
These cells have a short lifespan and cannot survive long in culture.
3. Preparation of Myeloma Cells
Myeloma cells are cancerous plasma cells.
Characteristics
- Grow continuously in culture
- Do not produce antibodies
- HGPRT deficient
These cells alone cannot survive in HAT medium.
4. Cell Fusion
Spleen cells and myeloma cells are mixed together.
Fusion is induced by:
- Polyethylene glycol (PEG)
or
- Sendai virus
After fusion, three types of cells are formed:
- Unfused spleen cells
- Unfused myeloma cells
- Hybridoma cells
5. Selection in HAT Medium
The mixture is cultured in HAT medium containing:
- Hypoxanthine
- Aminopterin
- Thymidine
Importance of HAT Medium
- Unfused spleen cells: Die naturally after some time
- Unfused myeloma cells: Cannot survive because they lack HGPRT enzyme
- Hybridoma cells: Survive because they obtain HGPRT enzyme from spleen cells
- Also possess immortality from myeloma cells
6. Screening of Hybridoma Cells
- Not all hybridomas produce the required antibody.
- Therefore, screening is performed using techniques such as:
- ELISA
- Radioimmunoassay
- Immunofluorescence
- Desired antibody-producing hybridomas are selected.
7. Cloning of Hybridoma Cells
- Selected hybridoma cells are cloned to ensure:
- Monoclonality
- Pure antibody production
- Methods include:
- Limiting dilution method
- Soft agar cloning
8. Production of Monoclonal Antibodies
- Large-scale production is carried out by:
- Tissue culture methods
or - Ascitic fluid method in mice
- The antibodies are then purified and stored.
Diagrammatic Representation
- Antigen injected into mouse
- Spleen cells isolated
- Myeloma cells prepared
- Cell fusion with PEG
- HAT medium selection
- Screening of hybridomas
- Cloning
- Monoclonal antibody production
Human Monoclonal Antibodies
Initially, monoclonal antibodies were produced entirely from mouse cells.
These mouse antibodies caused several problems in humans such as:
- Immune reactions
- Reduced effectiveness
Rapid destruction in body
- To overcome these limitations, human monoclonal antibodies were developed.
Types of Human Monoclonal Antibodies
1. Murine Antibodies
- These are completely mouse-derived antibodies.
Example
- Muromonab-CD3
- Problems
- Highly immunogenic
- Human anti-mouse antibody (HAMA) response occurs
Suffix:
- “-omab”
2. Chimeric Monoclonal Antibodies
These contain:
- Mouse variable region
- Human constant region
- About 65–70% human.
Advantages
- Less immunogenic
- Better tolerated
Example
- Rituximab
Suffix:
- “-ximab”
3. Humanized Monoclonal Antibodies
- Only antigen-binding regions are mouse-derived.
- More than 90% human.
Advantages
- Very low immunogenicity
- Longer half-life
Example
- Trastuzumab
Suffix:
- “-zumab”
4. Fully Human Monoclonal Antibodies
- These are completely human antibodies produced using:
- Recombinant DNA technology
- Transgenic mice
- Phage display techniques
Advantages
- Minimal immune reaction
- Highly effective
Example
- Adalimumab
Suffix:
- “-umab”
Production of Human Monoclonal Antibodies
1. Recombinant DNA Technology: Genes encoding antibodies are manipulated in laboratories to produce desired antibodies.
2. Phage Display Technology
-
- Antibody genes are inserted into bacteriophages.
- Phages displaying specific antibodies are selected.
3. Transgenic Mice Technology
-
- Mice are genetically engineered to contain human immunoglobulin genes.
- When immunized, they produce human antibodies.
Applications of Monoclonal Antibodies
Monoclonal antibodies have enormous applications in medicine, research, and industry.
1. Diagnostic Applications
A. Pregnancy Test
- Detection of human chorionic gonadotropin (hCG) in urine.
- Widely used home pregnancy kits use monoclonal antibodies.
B. Detection of Infectious Diseases
- Used for diagnosis of:
- HIV
- Hepatitis
- Dengue
- COVID-19
- Malaria
C. Cancer Diagnosis
- Monoclonal antibodies detect tumor markers such as:
- PSA
- AFP
- CEA
D. Blood Grouping
- Used for accurate identification of blood groups.
E. Hormone Assays
- Used for estimation of:
- TSH
- Insulin
- LH
- FSH
2. Therapeutic Applications
A. Cancer Therapy
- Monoclonal antibodies target cancer cells specifically.
Examples
- Rituximab for lymphoma
- Trastuzumab for breast cancer
- Bevacizumab for colon cancer
Mechanisms
- Direct killing of tumor cells
- Activation of immune system
- Delivery of toxins/radioisotopes
B. Autoimmune Diseases
- Used in:
- Rheumatoid arthritis
- Psoriasis
- Crohn disease
Example
- Adalimumab
C. Organ Transplantation
- Prevent transplant rejection by suppressing immune response.
Example
- Basiliximab
D. Allergic Disorders
Example
- Omalizumab for asthma
- E. Infectious Diseases
Used against:
- RSV infection
- Ebola virus
- COVID-19
3. Research Applications
- Monoclonal antibodies are important research tools.
Used in:
- ELISA
- Western blot
- Immunofluorescence
- Flow cytometry
- Cell marker identification
4. Imaging Techniques
- Radio-labelled monoclonal antibodies help in locating tumors inside the body.
- This technique is called immunoscintigraphy.
Advantages
- Highly specific
- Minimal cross-reaction
- Large-scale production possible
- Consistent quality
- Useful in targeted therapy
- High sensitivity in diagnosis
Limitations
- Expensive production
- Time-consuming process
- Possible immune reactions
- Limited penetration into tissues
- Resistance may develop
Recent Advances
- Modern biotechnology has greatly improved monoclonal antibody production.
- Important Advances
- Bispecific antibodies
- Antibody-drug conjugates
- Humanized antibodies
- Nanobodies
- CAR-T cell therapy association