Monoclonal Antibodies (mAb): Principle, Procedure, Types and Applications

Monoclonal antibodies (mAb) are defined as the antibodies derived from a single clone of plasma cell; all having the same antigen specificity, i.e. produced against a single epitope of an antigen.

Polyclonal vs Monoclonal Antibodies  

When an antigen having multiple epitopes enters the body, each epitope may stimulate one clone of B cells producing one type of antibody. Hence the resultant antibody mixture present in serum is polyclonal in nature, i.e. contains mixture of antibodies derived from different clones of B cells. Because one organism contains many different epitopes, the host’s serum contains various polyclonal antibodies.

Polyclonal antibodies used in immunodiagnosis are prepared by immunizing animals (usually rabbits, sheep, or goats) with an infectious agent and then isolating and purifying the resulting antibodies from the animal’s serum.

However, when only one clone of B cell is stimulated by a single epitope of an antigen and then is allowed to proliferate and produce antibodies; such antibodies are referred to as monoclonal antibodies (mAb).  Monoclonal antibodies are produced by Hybridoma technique, developed by G Kohler and C Milstein (1975), for which they were awarded Nobel Prize in 1984.

Principle

A clone of B cell stimulated against a single epitope of antigen (i.e. antibody-producing plasma B cell) is fused with malignant antibody-producing myeloma cell to produce a hybridoma cell. This hybridoma cell has two unique properties:

  1. Produces monoclonal antibody of same antigen specificity (due to B cell component).
  2. Multiplies indefinitely producing a clone of identical cells (due to immortal myeloma cell component).

Procedure

The process starts by immunizing a mouse with the antigen for which an antibody is to be produced.

Production of monoclonal antibody

Mouse splenic B cells: The mouse is injected with an antigen containing the desired epitope. The animal responds by producing many antibodies to the epitope injected.  After an interval, the mouse’s spleen which contains antibody-producing plasma cells is removed and emulsified so that antibody-producing cells can be separated and placed into individual wells of a microdilution tray. These cells cannot remain viable in the cell culture for a long time, so they must be fused with cells that are capable of surviving and multiplying in tissue culture.

Myeloma cells are used as a source of such immortal cells. They are cancerous plasma cells. They closely resemble mouse B cells; hence are compatible for fusion. However, myeloma cells also have the capacity to produce their own antibodies. Hence myeloma cells are genetically modified with two mutations (double mutated myeloma cells) so that they lose the ability to produce their own antibody but retain immortal property.  These cells are also deficient in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). This defect leads to their inabilities to survive in a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium).

Fusion: The mouse splenic B cells and mutated myeloma cells are fused in polyethylene glycol broth. In the reaction chamber, as a result, three types of cells are generated:

  1. Unfused myeloma cells
  2. Unfused mouse splenic B cells
  3. Fused hybridoma cells

Purification (by subculturing on HAT media):

The next step is to remove the unwanted unfused cells and to propagate the clone of hybridoma cells. This is carried out by subculturing the cells in reaction chamber onto a special medium called HAT medium (medium containing hypoxanthine, aminopterin and thymidine).

  • Mammalian cell (e.g. splenic B cell) synthesize purine by either de novo or salvage pathways.
  • Aminopterin blocks the de novo pathway so that the cell has to perform the salvage pathway to synthesize purines for its survival.
  • Salvage pathway requires two important enzymes- HGPRT and thymidine kinase.

As myeloma cell lacks HGPRT, it cannot grow on HAT medium but antibody- producing spleen cells can survive as they possess this enzyme.

Fate of three types of cells on HAT media:

  1. Hybridoma cells: Fused hybridoma cells survive in the selective medium and can be recognized by their ability to grow indefinitely in the medium.
  2. Unfused splenic B cells: They can grow, but do not survive long as they are not immortal.
  3. Unfused myeloma cells: They cannot grow as they lack HGPRT enzyme to perform the salvage pathway of purine synthesis.

If the original antigen used has multiple epitopes, many B cells would fuse with myeloma cells to produce a mixture of hybridoma cells each having specificity for one epitope. The medium containing hybridoma cells is then diluted into multi-well plates to such an extent that each well contains only one cell. The growth medium supernatant from the microdilution tray wells in which hybridoma cells are growing is then tested for the presence of desired monoclonal antibody using radioimmunoassay or ELISA techniques. 

When a good candidate antibody-producing cell is found, the hybridoma cells are selectively proliferated either in cell culture in vitro or they are reinjected into the peritoneal cavity of many mice, where the cells multiply and produce large quantities of antibody in the ascitic (peritoneal) fluid. Ascitic fluid can be removed from mice many times over the animals’ lifetimes, and the desired antibody is harvested. Such mAb may not be in pure form (maybe mixed with other antibodies); hence, it is purified by chromatography or by immunoprecipitation test.

Types of Monoclonal Antibodies

The above-mentioned procedure would yield monoclonal antibodies whose 100% amino acids are mouse-derived. The problem of mouse monoclonal antibody is that the mouse proteins being foreign; can induce immune response in humans producing human anti-mouse antibodies (HAMA); that in turn eliminate the monoclonal antibodies faster from the body. Hence mouse-derived monoclonal antibodies are not the best for human use.

Since the discovery of hybridoma technique, various modifications have been attempted to produce monoclonal antibodies by recombining human and mouse proteins.

Types of monoclonal antibodies
  1. Mouse mАb (-omab): It contains 100% mouse-derived proteins. They can lead to an allergic reaction in humans.
  2. Chimeric mAb (-ximab): It is prepared by recombination of mouse proteins (variable region) and human proteins (constant region). These can also cause an allergy.
  3. Humanized mAb(-zumab): Here, only the antigen-binding site (i.e. CDR-complementarity determining region) is mouse derived (10%) and the remaining part of mAb is human-derived.
  4. Human mAb (-umab): It contains 100% human-derived amino acids. It is the best-accepted mAb in humans.
Common Antibody Origin INN Substem Representative Examples
Chimeric -xi- Abciximab, Rituximab, Infliximab, Cetuximab
Humanized -zu- Palivizumab, Trastuzumab, Bevacizumab, Natalizumab
Human -u- Adalimumab, Panitumumab Golimumab, Ipilimumab

Applications of Monoclonal Antibodies

Isolation and purification: Monoclonal antibodies can be used to purify individual molecule from a mixture even when they are present in low concentration, e.g. interferon and coagulation factor VIII.

Identification of cells and clones: For example TH, and TC cells are identified by using anti-CD4 and anti-CD8 mAb.

Diagnostic reagents: The antigen detection kits employ various mAb tagged with detection molecules, such as fluorescent dye or enzyme to detect the specific antigens in the clinical specimen such as:

  • Detection of infections, such as hepatitis B, serogrouping of streptococci, etc.
  • Pregnancy detection test-by using monoclonal antibody against human chorionic gonadotropin.
  • Blood grouping can be done by using anti-A and anti-B monoclonal antibodies.
  • Tumor detection and imaging: By using mAb specific for tumor antigens secreted by tumor cells (e.g. prostate-specific antigen).
  • Tissue typing for transplantation can be done by using anti-HLA monoclonal antibodies.

Monitoring proteins and drug levels in serum.

Passive immunity: For post-exposure prophylaxis against various infections, mAb targeting specific antigens of the infecting organism can be administered. Examples include-immunoglobulins against hepatitis B, rabies, and tetanus.

Therapeutic use: Monoclonal antibodies are used in the treatment of various inflammatory and allergic diseases and cancers. Monoclonal antibodies (mAbs) are useful to treat some of the cancer types. Naked mAbs (antibodies without attached drug or radioactive material) are the most common type of mAbs used to treat cancer. So far, the US FDA has approved more than a dozen mAbs e.g. alemtuzumab, trastuzumab to treat certain cancers. Similarly, basiliximab is used to treat transplant rejection while belimumab treats systemic lupus erythematosus.

You can check the list of FDA approved monoclonal antibodies are their use in this link.
The mechanisms by which the mAb work as a therapeutic agent are:

  • Suppress the immune system
  • Kill or inhibit malignant cells
  • Inhibit angiogenesis.

Used as immunotoxin: mAb conjugated with bacterial/ chemical toxins (e.g. diphtheria toxin) can be used to kill the target cells such as cancer cells. Here, mAb against surface receptors helps in binding to the target cells and the toxin helps in target cell killing.

Used as enzymes: Abzyme is a monoclonal antibody with catalytic activity.

References and further readings

About Acharya Tankeshwar 403 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

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