Which of the following are TRUE statements concerning HAT selection? HAT medium

Question

Which of the following are TRUE statements concerning HAT selection? HAT medium selection is used to generate a polyclonal antibody Myoloma cells are killed as they are mortal HAT selection requires the fusion of helper T cells with myoloma cells HAT medium selection is used to generate a monoclonal antibody You isolate only Hybridoma clones that recognize your specific antigen from HAT selectio Myoloma cells die as they cannot synthesize new nucleotides HAT selection requires the fusion of plasma cells with myoloma cells

Explanation / Answer

1. False. HAT(Hypoxanthine aminopterin thymidine) medium is a selective medium usually used for the production of monoclonal antibodies through a process known as Hybridoma technology. For this purpose, a mouse is first immunized with an antigen of interest which further leads to the proliferation of antigen-specific B cell and thereby antibody generation. Then B cells are isolated from the spleen of mouse and fused with myeloma cells lacking HGPRT(Hypoxanthine Guanine Ribosyl Transferase). Now considering the aminopterin present in the HAT medium is responsible for inhibiting the de novo nucleotide synthesis pathway by blocking the tetrafolate which is crucial for this pathway, cells can also go for salvage pathway for nucleotide synthesis when the de novo pathway is affected.Here, in this case, cells are unable to utilize salvage pathway because of myeloma cells that are deficient in HGPRT(crucial enzyme for salvage pathway). As a consequence myeloma cells will die along with unfused B cell as they have a short lifespan. Hybridomas are selected owing to the HAT medium which allows the hybridoma having characteristic from both fusion partners(that is an ability to produce antibodies and immortal) to survive and kills unfused myeloma cells.Such antibody has identical isotype and single specificity and therefore monoclonal antibodies.

2.False. Myeloma cells are immortal, they are capable of dividing indefinitely. Only unfused myeloma cells lacking HGPRT will die as they cannot synthesise nucleotide via salvage pathway when the de novo pathway is already disrupted by aminopterin present in HAT medium.

3. False. HAT selection medium requires the fusion of B cells with myeloma cells.

4.True.

5.True.

6.True.

7.True.

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