A Technique to Generate Monoclonal Antibodies from Antigen-Specific B Cells
Published: March 29, 2024
Abstract
Source: Devilder, M., et al. Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood. J. Vis. Exp. (2018).
The video demonstrates a technique for generating monoclonal antibodies from antigen-specific B cells. Eukaryotic expression vectors encoding the heavy and light chains of antibodies, cloned from isolated antigen-specific B cells, are introduced into mammalian cells using polyplexes. Once inside the cells, these vectors undergo translation, and the resulting heavy and light chain peptides assemble into antibodies, which are subsequently secreted by the cells.
Protocol
1. Production of mAbs The day before the transfection, seed 15,000 Human embryonic kidney 293A (HEK 293A) cells in 200 µL of Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS per well in 96-well plates. Incubate overnight in a CO2 incubator (5% CO2) at 37 °C. Cotransfect 293A cells in DMEM medium containing 10% FBS using a linear polyethylenimine derivative as transfection reagent. NOTE: Perform the transfec…
Offenlegungen
The authors have nothing to disclose.
Materials
| HEK 293A cell line | Thermo Fisher scientific | R70507 | |
| DMEM (1X) Dulbecco's Modified Eagle Medium | Gibco by life technologies | 21969-035 | (+) 4,5g/L D-Glucose 0,11g/L Sodium Pyruvate (-) L-Glutmine |
| Nutridoma-SP | Roche | 11011375001 | 100X Conc |
| Fetal Bovine serum (FBS) | Dominique Dutscher | S1810-500 | |
| Jet PEI DNA transfection reagent | PolyPlus | 101-40 | |
| Flat bottom96-well plate | Falcon | 353072 | |
| V-bottom 96-well plate | Nunc/Thermofisher | 55142 |
Tags
Diesen Artikel zitieren
A Technique to Generate Monoclonal Antibodies from Antigen-Specific B Cells. J. Vis. Exp. (Pending Publication), e22090, doi: (2024).