A Technique to Generate Monoclonal Antibodies from Antigen-Specific B Cells

Samenvatting

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

1. 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.
2. Cotransfect 293A cells in DMEM medium containing 10% FBS using a linear polyethylenimine derivative as transfection reagent.
   NOTE: Perform the transfection in triplicates. The following protocol is for one well of a flat bottom 96-well plate.
   1. Dilute 0.5 µL of DNA transfection reagent int....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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