Generating Recombinant Monoclonal Antibodies From Mammalian Cell Cultures
Transcrição
Prepare a transfection mix by adding an appropriate cationic polymer and separate plasmid vectors for heavy-chain and light-chain antibody genes.
The positively charged polymer binds with the negatively charged plasmids, forming complexes.
Add this transfection mix to a multi-well plate containing human embryonic kidney cell culture in a serum-free medium and incubate.
The complexes enter the cells through endocytosis, causing endosomal swelling and rupture, releasing them into the cytoplasm.
The cationic polymer produces pores in the nuclear membrane, facilitating vector entry and enabling their transcription, followed by translation into heavy- and light-chain proteins.
These proteins move to the endoplasmic reticulum, where heavy- and light-chain proteins are dimerized and covalently linked by disulfide bonds, forming antibodies.
Antibodies travel to the Golgi apparatus for additional post-translational modifications and are packaged into vesicles for extracellular release.
Collect the medium containing the released antibodies. Add a serum-free medium for continued antibody generation.
