1. HYBRIDOMA TECHNOLOGY

2. Hybridoma technology for the production of monoclonal antibodies (MABs) has contributed significantly to aquaculture.
Monoclonal antibodies are being employed in disease, pathogen classification, epidemiological analysis and development of vaccines.
Hybridoma technology is a technology of forming hybrid cell lines (called hybridomas) by fusing a specific antibody-producing B cell with a myeloma (B cell cancer) cell that is selected for its ability to grow in tissue culture.
The antibodies produced by the hybridoma are all of a single specificity and are therefore monoclonal antibodies (in contrast to  polyclonal antibodies ).

3. The idea of a “magic bullet” was first proposed by  Paul Ehrlich.
In the 1970s the B-cell cancer multiple myeloma was known, and it was understood that these cancerous B-cells all produce a single type of antibody.
This was used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a given  antigen.

4. Production of monoclonal antibodies involving human–mouse hybrid cells was described by Jerrold Schwaber in 1973. 
The invention was conceived by Prof. Pieczenik, with Prof. John Sedat, and produced by Kohler and Milstein.
Kohler, Milstein and Jerne in 1975; who shared the  Nobel Prize in Physilogy or Medicine in 1984 for the discovery.
The key idea was to use a line of myeloma cells that had lost their ability to secrete antibodies, come up with a technique to fuse these cells with healthy antibody-producing B-cells, and be able to select for the successfully fused cells.

5. Production of monoclonal antibodies
MAbs production is initiated by the immunisation of BALB/c mice with immunogens.
Antibodies are produced by differentiated B-cells (plasma cells) of a particular specificity, the antibodies are identical and therefore, is a source of homologous antibodies.
Plasma cells are, however, short-lived and cannot be grown in culture.
Therefore, fusion of these cells with immortal myeloma cells produces hybridoma cells with the ability to grow in culture and to secrete antibody with a defined specificity.
Chemical selection, screening of the antibodies produced and cloning of the hybridoma cells lead to the ultimate production MAbs.

6. Monoclonal antibodies can be prepared against a wide variety of immunogens, e.g., protein, carbohydrate, nucleic acid or combinations of these.
They can also be produced from impure antigen by selecting single cell clone after the fusion.
The resulting MAbs are extremely specific and are therefore, very useful diagnostic tools.
In addition, hybridoma cell lines have the advantage of providing an unlimited supply of the antibody in the cell supernatant, which allows standardisation of the MAb reagents.

7. Myeloma cell lines used in fusions have been selected because they do not produce antibody molecules, although some of the commercially available cell lines do produce immunoglobulin heavy or light chain molecules.
For this reason P3x63. Ag8-653 (653) and Sp2/0-Ag14 (Sp2/0) are the most frequently used cell lines in hybridoma technology.
Hybridoma cells can be prepared by fusing myeloma cells and antibody – producing cells which have been isolated from different mouse species.
But the success rate of fusion is greatly increased if both cell types come from the same strain of mouse (e.g., BALB/c).

8. Kohler and Milstein used Sendai virus as the fusion agent.
Polyethylene glycol (PEG) is now routinely used to fuse the cells.
Even in efficient fusions, only approximately 1% of the initial cell numbers result in fusion.
This leaves a large number of unfused cells, both spleen and myeloma cells still present in the culture.
The spleen cells from the mouse die within 3 days of culture and therefore, do not pose a problem.
However, the myeloma cells quickly adapt to the culture conditions and will outgrow the hybridoma cells resulting from the fusion.

9. Removal of the myeloma cells is therefore, essential and is achieved by chemical selection.
Commercially available myeloma cells are defective in one of the enzymes of the salvage pathway of purine nucleotide biosynthesis.
Cell lines 653 and SP2 have mutations of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene.
Addition of aminopterin to the culture medium blocks the de novo nucleotide synthesis pathway and forces the cell to use the salvage pathway in which HGPRT uses exogenous hypoxanthine and thymidine.
Myeloma cells defective in HGPRT are unable to use this pathway and therefore, die in culture.

10. The only cells able to grow in HAT (hypoxanthine, aminopterin, thymidine) culture medium are the hybridoma cells, which are unable to synthesize DNA via de novo nucleotide synthetase pathway and rely on the salvage pathway for DNA synthesis (a characteristic provided by the spleen cell part of the hybridoma).
Positive clones producing specific antibodies are usually identified by ELISA and are selected, expanded and cloned using a limiting dilution technique. Positive hybridomas are normally cloned three times before they are considered MAb producing cells.

11. Application of Monoclonal Antibodies in Fish Farming
Our knowledge of the immune system of fish and fish diseases is extremely limited when compared to our knowledge of large animals.
At present, fish farming is becoming an increasingly important food production industry, and may play a significant role as a food source in the future.
For this reason, application of the latest biotechnological advances, including MAbs, to the aquaculture industry, is extremely important.
MAbs are being adopted for purposes of immunoassay and immunotherapy.

12. Though the technology for MAb production has been in existence for more than 25 years, yet this application to fish farming is still in its infancy.
Today, monoclonal antibodies to several viral and bacterial pathogens of fish and shellfish are available in the market.
It has been possible to develop rapid, simple, cheap, specific and sensitive MAb based immunodiagnostic kits for several microbial pathogens.
MAb based diagnostic kits such as ELISA and immunodot have even been simplified to the field level for use by farmers.
Furthermore, detection of minute serological difference among bacterial and viral variants of fish and shellfish is possible by MAb based epitope analysis.
This has helped immensely in serological and epidemiological studies.

13. Monoclonal antibodies were produced against enterotoxin of Vibrio cholerae, a brackishwater and estuarine bacterium which causes cholera.
MAbs based ELISAs have been used for studies of Vibrio anguillarum strains and for rapid diagnosis of clinical cases of Enteric Red mouth (Yersinia ruckeri) and furunculosis (Aeromonas salmonicidae) in fish farms.
MAbs are also used to study piscine parasities.
MAbs have been developed against Bonamia ostreae, Ceratomyxa shastia, Cryptobia salmonsitica, Perkinsus maximus are pathogenic protozoan of shell fish.

14. MAbs have also been employed for analysis of lymphocyte receptors and characterization of lymphocyte population in carp,
for immunopurification of salmon prolactin and for development of sandwich ELISA system for both salmon prolactin and somatotropin.
MAbs to A. hydrophila, EUS fungus Aphanomyces invadans and white spot virus of shrimp have been produced and being used in diagnosis in India.
The use of monoclonal antibody techniques would prove extremely useful in the development of diagnostic tests for certain important diseases of fish, whose incidence is likely to increase with the higher stocking densities necessary for successful fish farming.

15. 1. E.g. Detection of Infectious Pancreatic Necrosis virus ( IPNV ) by ELISA. ELISA could be used for the identification of different serotype of IPNV.
2. Application of a MAb against virus:- Infectious hematopoietic necrosis (IHN), caused by IHN virus (IHNV), is a severe and acute epizootic among salmonid fish. This disease is now widespread. MAbs against IHNV HV , were produced.

16. Thank you