Production and Purification of Recombinant Antibodies from Mammalian Cells
Published: March 29, 2024
Abstract
Source: Shivange, Gururaj, et al. Analyzing Tumor and Tissue Distribution of Target Antigen-Specific Therapeutic Antibody. J. Vis. Exp.(2020).
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…
Offenlegungen
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 | Authenticated by STR profiling |
Tags
Diesen Artikel zitieren
Production and Purification of Recombinant Antibodies from Mammalian Cells. J. Vis. Exp. (Pending Publication), e22082, doi: (2024).