Table of Contents
Why hypoxanthine aminopterin and thymidine HAT medium was used in the production of monoclonal antibodies?
The trick is that aminopterin blocks DNA de novo synthesis, which is absolutely required for cell division to proceed, but hypoxanthine and thymidine provide cells with the raw material to evade the blockage (the “salvage pathway”), provided that they have the right enzymes, which means having functioning copies of the …
What is the use of aminopterin in hybridoma production?
Aminopterin is a dihydrofolate reductase inhibitor: treatment of cells with aminopterin prevents de novo DNA synthesis, and in the absence of exogenous hypoxanthine and thymidine to supply the salvage pathway, the cells will die.
How does aminopterin block de novo synthesis?
The aminopterin found in the medium blocks the de novo DNA nucleotide synthesis pathway. Typically when the de novo pathway is blocked, cells will then utilize the salvage pathway as an alternative means to replicate (only if hypoxanthine and thymidine are present).
Why are myeloma cells used in monoclonal antibodies?
Hybridoma technology is a method for producing large numbers of identical antibodies (also called monoclonal antibodies). The myeloma cell line that is used in this process is selected for its ability to grow in tissue culture and for an absence of antibody synthesis.
What is the purpose of HAT medium in the preparation of monoclonal antibody?
The purpose of the medium is to: (1) selectively kill unfused myeloma cells that are well adapted to tissue culture and would otherwise outgrow any hybridomas produced and (2) eliminate any myeloma-myeloma hybridomas that lack HPRTase.
Why only hybridoma cells survive in the selection medium during the production of the monoclonal antibody?
Removal of the unfused myeloma cells is necessary, because they are potentially capable of outgrowing other cells, especially weakly established hybridomas. Unfused B cells also die as they have a short life span. Thus, only the B cell-myeloma hybrids survive because the HGPRT gene in B cells is functional.
What is a aminopterin used for?
Aminopterin is an amino derivative of folic acid, which was once used as an antineoplastic agent in the treatment of pediatric leukemia. In the 1950’s its production was discontinued in favor of methotrexate, which is less potent but less toxic. Off label, aminopterin has also been used in the treatment of psoriasis.
What is the function of aminopterin?
Aminopterin works as an enzyme inhibitor by competing for the folate binding site of the enzyme dihydrofolate reductase. Its binding affinity for dihydrofolate reductase effectively blocks tetrahydrofolate synthesis.
Why are Tumour cells used to make monoclonal antibodies?
Some monoclonal antibodies block the connection between a cancer cell and proteins that promote cell growth — an activity that is necessary for cancer growth and survival. Preventing blood vessel growth. In order for a cancerous tumor to grow and survive, it needs a blood supply.
What is the purpose of using myeloma cells?
Myeloma cells prevent the normal production of antibodies, leaving your body’s immune system weakened and susceptible to infection. The multiplication of myeloma cells also interferes with the normal production and function of red and white blood cells.
What is the function of hat?
A hat is a head covering which is worn for various reasons, including protection against weather conditions, ceremonial reasons such as university graduation, religious reasons, safety, or as a fashion accessory. In the past, hats were an indicator of social status.
How the hybridoma cells are selected?
Hybridomas are thus selected by continuously feeding the mixture of the three different cell types with HAT medium (kept in 96-well microtiter plates in a carbon dioxide incubator at 37°C): Unfused plasma cells are easily eliminated in these conditions since they do not replicate in culture medium.