The why, how and what to do of generating your own antibody. Basics and practical aspects.

The mechanism of immune response lies with two key elements called as antibodies and antigens. Immune system of an organism depends on recognition of a molecule which is foreign. The B lymphocytes play an active role in production of antibodies. The antibodies are specific to specific antigen. Immunogenic specificity is a key role in the production of specific antibodies which are specific to individual epitope on immunogenic surface.
There are basically two types of antibodies namely monoclonal antibodies and polyclonal antibodies. Polyclonal antibodies are generated from B-lymphocytes or clones, which are of multiple origins. These antibodies bind to different epitopes and they are obtained from serum of immunized animal. Monoclonal antibodies are generated from specific epitope on B lymphocytes. The preliminary step is crucial which involves animal selection. The recommended species involved in production of antibodies are rabbit, goat, and chicken.
The production of monoclonal antibodies involves in vivo and in vitro steps. The procedure basically involves a cell line, which is capable of producing antibodies and an immortal cell line called as hybridoma. The hybridoma technology is used for the production of monoclonal antibodies. The steps involved are
1. Selection of donors for hybridoma cell generation and immunization: The animal is immunized with immunogen that is prepared either by adjuvants or emulsifying the antigen with Freund’s adjuvant imbibed with antigen. Antigens such as microorganisms, membranes, cells or synthetic peptides are frequently used. The immunization is done every 2-3 weeks until sufficient titer of antibodies is attained in the serum. The animals are then euthanized and spleen is removed to facilitate cell fusion with myeloma cells.
2. The blood samples obtained from the animal are measured for serum antibodies. The titer can be detected by enzyme-linked immunosorbent assay and flow cytometry. If the titer is satisfactory then cell fusion can be started otherwise booster doses are injected. Once a satisfactory level is achieved, spleen from the animal is harvested.
3. Myeloma cell preparation: The fusion of myeloma and antibody producing spleen cells results in hybridoma which is capable of unlimited growth. The myeloma cells are cultured in 8-azoguanineto to ensure that these cells are sensitive to hypoxanthine-aminopterin-thymidine selection medium. Hybridoma cells are highly viable and have proliferent growth. The important issue here is that only fused cells grow in HAT medium and rest die.
4. Polyethylene glycol is used to fuse myeloma cells and harvested spleen cells as PEG causes fusion of membranes. Macrophages are used as feeder cells for the hybridoma cells and they are produced from murine bone marrow.
The selection medium contains aminopterin, which is involved in blockade of synthetic pathways that produces nucleotides. The cells pass through alternative pathway for production of nucleic acids which is defective in myeloma cells and produce antibodies.