4. Disturbance of host physiology- if the gene integrate to a wrong place in the genome, it may cause functional defects. In few cases, it may disrupt the function of the tumor suppression genes results into the development of the tumor. For example, X-linked severe combined immunodeficiency (X-SCID) patients injected with the transformed hematopoietic stem cells led to the development of T-cell leukemia.

(C) MONOCLONAL ANTIBODY PRODUCTION

Antibodies are used in the adjuvant therapy to remove the infectious agents or toxic substances. Monoclonal antibodies are generated by the hybridoma technology. The complete process of hybridoma technology to produce antibody has seven steps (Figure 40.2).

STEP 1: Immunization of mouse- mouse is injected with the desired antigen to produce antibodies. It is performed by a series of injections over the course of several weeks.

STEP 2: Isolation of B-Cells- splenocytes are isolated from the spleen of the mouse.

STEP 3: Cultivation of Myeloma cells- Myeloma cells are grown in the serum containing complete media.

STEP 4: Fusion of myeloma and B-Cells- B-Cells and myeloma cells are fused using polyethylene glycol (PEG) or the Sendai virus.

STEP 5: Screening of fused cells- Fused cells are grown in the HAT medium (hypoxanthine-aminopterin-thymidine medium) for an extended period. Myeloma cells lacks salvage pathways, and they die in the presence of the aminopterin, an inhibitor blocks the nucleotide synthesis. The fused cells grow in HAT medium due to contribution of genetic material from B-cells. Fused hybridomas are screened to test the production of desirable antibody against the antigen. The culture supernatant is tested for the presence of antibody in a sandwich ELISA.

STEP 6: Multiplication- The hybridomas producing desirable antibody can be propagated in the medium containing IL-6 to establish hybridoma cell lines. It grows in culture medium to produce antibodies.

STEP 7: Harvesting- The hybridomas are transferred into the large culture flasks to secrete antibodies. The culture supernatant can be collected and stored at -200C or other lower temperature for future usage.