Encyclopedia of Experiments: Immunology
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Transcript
Take eukaryotic expression vectors, encoding either an antibody heavy chain or an antibody light chain, cloned from antigen-specific B cells.
Introduce polyethylenimine, a cationic polymer, that electrostatically interacts with the negatively-charged vectors, forming polyplexes.
Incubate mammalian cells with the polyplexes, which adhere to the cells and are internalized via endocytosis. The resulting endosomes fuse with lysosomes.
The polymers in the polyplexes cause osmotic swelling of the vesicle, leading to its rupture and release of the vectors.
The released vectors enter the nucleus and are transcribed into heavy-chain and light-chain mRNA, which undergo translation via ribosomes on the endoplasmic reticulum.
The resulting peptide chains assemble into antibodies, which are transported to the Golgi and packaged into secretory vesicles.
Upon fusion of the secretory vesicles and the cell membrane, the antibodies are released extracellularly.
Harvest the antibody-containing supernatant for downstream assays.
