Generating Recombinant Monoclonal Antibodies From Mammalian Cell Cultures

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.

Prepare the HEK cells in supplemented DMEM and grow them to 75% confluency in 75-square-centimeter culture flasks. For each heavy/light chain transfection, prepare a mix with 1.75 milliliters of DMEM without serum. Include 4.5 micrograms of heavy chain vector, 3 micrograms of light chain vector, and 50 microliters of polyethylenimine solution. Gently add this mixture to a plate of HEK cells and swirl the solution over the plate.

Incubate the plate overnight, and then begin collecting the culture media every day, replacing it with serum-less medium. The media contains the antibodies, and it can be collected daily for four days.