The video explains the process of producing antibodies in mammalian host cells, from gene expression to secretion. It involves transcription, translation, signal recognition, correct folding, and eventual release into the extracellular medium via secretory vesicles.
Protocol
1. Expression and purification of antibodies Maintenance of CHO cells Grow CHO cells in FreeStyle CHO Media supplemented with commercially available 1x glutamine supplement at 37 °C shaking at 130 rpm with 5% CO2 using delong Erlenmeyer flasks, either glass or disposable. NOTE: It is highly recommended to use a baffled flask with a vented cap for increased agitation and to improve gas transfer during shaking conditions. Sig…
Disclosures
The authors have nothing to disclose.
Materials
FreeStyle CHO media
Gibco Life Technologies
Cat # 12651-014
Anti-Anti (100X)
Gibco Life Technologies
Cat # 15240-062
Anti-Clumping Agent
Gibco Life Technologies
Cat # 01-0057DG
BD Insulin Syringe
BD BioSciences
Cat #329420
Caliper IVIS Spectrum
PerkinElmer
Cat #124262
CHO CD EfficientFeed B
Gibco Life Technologies
Cat #A10240-01
Corning 500 mL DMEM (Dulbecco's Modified Eagle's Medium)
Corning
Cat # 10-13-CV
Corning 500 mL RPMI 1640
Corning
Cat # 10-040-CV
Cy5 conjugated Anti-Human IgG (H+L)
Jackson ImmunoResearch
Cat # 709-175-149
GlutaMax-I (100X)
Gibco Life Technologies
Cat # 35050-061
HiPure Plasmid Maxiprep kit
Invitrogen
Cat # K21007
HiTrap MabSelect SuRe Column
GE Healthcare
Cat # 11-0034-93
Isoflurane, USP
Covetrus
Cat # 11695-6777-2
Lubricant Eye Ointment
Refresh Lacri-Lube
Cat #4089
PEI transfection reagent
Thermo Fisher
Cat # BMS1003A
Experimental Models: Cell lines
Human: OVCAR-3
American Type Culture Collection
ATCC HTB-161
Human: CHO-K cells
Stable transformed in our lab
ATCC CCL-61
Mouse: 4T1
Kind gift from Dr. Chip Landen, UVA
Mouse: MC38
Kind gift from Dr. Suzanne Ostrand-Rosenberg, UMBC