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…

Divulgazioni

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

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Citazione di questo articolo
A Technique to Generate Monoclonal Antibodies from Antigen-Specific B Cells. J. Vis. Exp. (Pending Publication), e22090, doi: (2024).

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