Question

Crohn’s disease is an inflammatory bowel disease most often localized to the lower part of the small intestines. It is thought that Crohn’s disease could be an autoimmune disease, because chronic inflammation and activated immune cells are found deep into the intestinal tissue of Crohn’s patients. The chronic inflammation causes the small intestines to swell, which can lead to abdominal pain, diarrhea, gastrointestinal bleeding, and/or weight loss (due to malnutrition), and if the inflammation is severe enough, the small intestine can swell shut and a blockage can ensue that needs to be surgically removed. Crohn’s disease is an uncomfortable syndrome that can lead to life-threatening events. A cytokine known as Interleukin-23 (IL-23) appears to play a role in Crohn’s disease. Mice injected with IL-23 develop inflammation in the small intestine similar to what is seen in Crohn’s disease in humans, and mice that are genetically deficient in IL-23 (known as IL-23 knockout mice) show reduced frequency of Crohn’s disease. Interesting! Upon looking further into the association between IL-23 and Crohn’s disease, you decide to create a monoclonal antibody that neutralizes the activity of human IL-23. Your rationale is that if too much IL-23 can cause Crohn’s disease, then an antibody that neutralizes IL-23 could be administered to Crohn’s patients and it might cure them of their disease. You obtain a vial of highly-purified human IL-23 from Dr. Louten. You intend to use it as an antigen for obtaining polyclonal antibodies from an animal.

Question 1: You know that mice only live about 1.5 to 2 years, so you wisely decide you need to make monoclonal antibodies against human IL-23. Starting with your mice that produce polyclonal antibodies, describe the process you would use to make immortalized hybridomas that produce monoclonal antibodies.

Answer 1

Antibody forming spleen cells are fused with myeloma cells to produce hybrid cells(hybridomas).The resultant hybridoma retains the antibody producing capacity of the spleen cell and ability of myeloma cells to multiply indefinitely.

The details of technique are as follows-

1) mouse is immunised with the desired antigen( here IL-23) and lymphocytes are harvested from the spleen.
2)spleen cells (lymphocytes) are fused with mouse myeloma cell grown in culture which are deficient in the enzyme hypoxanthine phosphoribosyl transferase.(HPRT). Fusion is done by incubating the cells in the presence of polyethylene glycol(PEG).
4) the fused cells( hybrid cells) are grown in basal culture medium containing hypoxanthine,amniopterin, thymidine (HAT medium).
5) only hybrid cells having properties of both the splenic lymphocytes (HPRT+) and myeloma cells (HPRT-) can grow in culture. the enzyme HPRT is necessary for nuclic acid synthesis and is provided by the splenic lymphocytes in hybrid cells. splenic lymphocytes alone cannot replicate indefinitely while unfuse myeloma cells are killed by amniopterin in HAT medium.
6) clones that ksecret the desired antibody are selected for continuous cultivation .this hybridomas can be maintained indefinitely and will continue to for monoclonal antibodies .They can also be grown as tumors in the peritoneal cavity of mice by intraperitoneal in inoculation and monoclonal antibodies are obtained by harvesting the ascitic fluid produced .hybridomas may be frozen for prolonged storage.