Encyclopedia of Experiments: Immunology
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Transcript
Take a mouse immunized with a metallopeptidase. Harvest the spleen and obtain a cell suspension.
Filter the suspension through a mesh and centrifuge, removing tissue debris.
The isolated cells include metallopeptidase-specific B cells, producing antibodies against metallopeptidase epitopes.
Add immortal myeloma cells lacking the HGPRT enzyme, preventing the salvaging of nucleobases for nucleotide synthesis. They survive via de novo nucleotide synthesis.
Centrifuge to obtain a pellet and discard the supernatant.
Add PEG and incubate, inducing myeloma-B cell fusion to form HGPRT-positive immortal hybridomas.
Add HAT medium comprising hypoxanthine, aminopterin, and thymidine. Transfer into wells containing a peritoneal macrophage feeder layer, promoting hybridoma growth.
Aminopterin blocks de novo nucleotide synthesis, causing unfused myeloma cells to die. Unfused B cells die due to their short lifespan.
The HGPRT-positive hybridomas salvage the nucleotide precursors hypoxanthine and thymidine, proliferate, and secrete metallopeptidase-specific monoclonal antibodies.
Harvest the supernatant to confirm antibody production via downstream assays.
