Monoclonal antibodies: Principle, Technique and Uses

• Antibodies are produced ordinarily by infection or immunization in an animal in response to a single antigen.
• They are heterogenous as they are synthesized by several different clones of antigens, i.e. they are polyclonal.
• Such antisera contain immunoglobulins of different classes with specificities against different epitopes of the antigen.

Principle

• Monoclonal antibodies are those which are produced by a single antibody forming cell or clone directed against a single antigen or antigenic determinant.
• Plasma cell tumour (myeloma) is one of its examples.
• In myeloma, antibodies are produced by a single clone of plasma cells.
• These are directed against a single antigenic determinant and hence the antibodies are homogenous and monoclonal.

• Monoclonal antibodies are very useful tools in diagnosis, and treatment of many diseases and also in research technology.
• Kohler and Millstein in 1975 prepared a hybrid cell line (hybridoma) by fusion of a mouse myeloma cell with an antibody producing lymphocyte by laboratory manipulation.
• This lymphocyte was taken from spleen or lymph node of the same inbred strain of mouse.
• Such hybrid cells can produce virtually unlimited quantities of monoclonal antibody of any required specificity indefinitely in cell culture conditions.
• For this technological revolution, they were awarded the Nobel Prize in 1984.

Technique

• The term hybrid refers to a diploid organism or cell which results from a cross between two genetically dissimilar parents or cells.
• Animal (usually mouse) is immunized with a pure selective antigen and when good immune response occurs, it is killed.
• B lymphocytes are harvested from the spleen or lymph node of this mouse.
• A suspension of spleen cells (B cells) is prepared. Further processes that are followed are:

1. Mouse myeloma cells are grown in a tissue culture medium deficient in the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT).
2. Myeloma cells are chosen as it can live indefinitely in culture and can produce huge quantities of immunoglobulin.
3. A suspension of splenic cells is then fused with a myeloma cell-line by incubating in presence of polyethylene glycol.
4. Un-fused myeloma and spleen cells are removed.
5. The few cells that have fused (hybrid cells) are grown in selective culture medium containing hypoxanthine-aminopterin and thymidine (HAT) in a number of wells for 10-14 days.
6. Such tissues cultures are called hybridomas or “immortal clones of cells” capable of producing monoclonal antibody.
7. The culture medium does not support the growth of enzyme deficient parent myeloma cell and normal B lymphocytes.
8. Only hybrid cells can grow in the selective medium.
9. After the growth of hybrid cells, the supernatant of each well are then tested for antigenic activity (by RIA, ELISA) and clones that secrete the desired antibody are selected.
10. When good candidate antibody-producing cell is found, the hybrid cells are grown in bulk for production of monoclonal antibody.
11. This is done either by culturing in large numbers in cell cultures in vitro or by injecting them in the peritoneal cavities of many mice.
12. Then they grow as tumors and produce large quantities of antibodies in the ascitic fluid.
13. The ascitic fluid can be aspirated from mice many times over the animal life time.
14. As all the cells are derived from a single type of hybrid cell, they produce only one type of antibody.
15. Such antibody is called monoclonal antibody.
16. The hybrid cells can be maintained in culture definitely.
17. Hybridoma can be frozen for prolonged storage.

Characteristics of hybrid cells

• Hybridomas are generally produced from special myeloma cell lines which carry two mutational defects:

i) Hybrid cells no longer produce their own immunolglobulin but that of the normal B cell parent.

ii) The hybrid cells lack the enzyme HGPRT necessary for nucleic acid synthesis which is provided by the B cell component of hybridoma.

Properties of Hybridoma

1. The hybrid cells have got capacity for limitless growth (immortality).
2. They can synthesize unlimited amount of Ig (10,000 molecules/cell/min).

• In addition to cell culture and mouse hybridomas, inter-species (rat-mouse and human-mouse) hybridomas have been developed.
• Recently human to human hybridomas have also been generated.

Uses of monoclonal antibodies

A) Diagnostic use

• Commercial diagnosing test kits for identification of bacterial, viral and other antigens, which includes hormones (HCG, progesterone, growth hormone, etc.), tumour associated antigen (leukaemia, lymphoma, carcinoma, sarcoma) and miscellaneous antigens (HLA-A/B/C/DR).

B)Test for vaccines

• Identification and purification of microbial products, both for vaccine and for industrial use.

C)Pure antibody

• Large amount of pure antibody of defined class and subclass can be prepared.

D)Future use

• Hybridomas can be stored in frozen state and revived subsequently.

*Murine antibodies are unsuitable for human therapeutic use because of strong immune response induced by them in human.

*In addition, the Fc piece of mouse Ig fails to initiate effector defense mechanisms in human.

* Human monoclonal antibodies are now being prepared by fusion of particular antibody fragment genes to bacteriophage genes.

*Thus, large quantities of desired monoclonal antibody can be prepared by infecting bacteria with appropriate bacteriophage.

References:

i) https://www.mayoclinic.org/diseases-conditions/cancer/in-depth/monoclonal-antibody/art-20047808

ii) https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/monoclonal-antibody

iii) https://www.britannica.com/science/monoclonal-antibody

Monoclonal antibodies: Principle, Technique and Uses