a technique to generate monoclonal antibodies from antigen-specific b cells

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

Seed 15,000 human embryonic kidney cells in 200 microliters of Dulbecco&#39;s Modified Eagle Medium supplemented with 10% fetal bovine serum in each well of a flat-bottom 96-well plate, a day before the transfection. Then, incubate the plate at 37 degrees Celsius overnight at 5% carbon dioxide. Cotransfect the cells using a linear polyethyleneimine derivative transfection reagent. Dilute 0.5 microliters of DNA transfection reagent with 10 microliters of 150 millimolar of sodium chloride.
Next, dilute 0.125 micrograms of each variable heavy and light chain-expressing vectors in 10 microliters of 150 millimolar sodium chloride. Vortex all the dilutions for 10 seconds each. Then, mix the previously diluted DNA transfection reagent with 10 microliters of DNA solution. Quickly vortex the DNA transfection mixture for 15 seconds, and incubate for 15 minutes at room temperature.
Add the DNA transfection mixture dropwise on the cells, and then, gently swirl the plate. Replace the medium 16 hours after transfection, and culture the cells for five days in serum-free medium at 37 degrees Celsius.

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1. Production of mAbs
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	<li>The day before the transfection, seed 15,000 Human embryonic kidney 293A (HEK 293A) cells in 200 &#181;L of Dulbecco&#39;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 &#176;C.</li>
	<li>Cotransfect 293A cells in DMEM medium containing 10% FBS using a linear polyethylenimine derivative as transfection reagent. 
	NOTE: Perform the transfec...

Source: Devilder, M., et al. <a href="https://app.jove.com/v/56508">Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood.</a> 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.

1. Production of mAbs

	The day before the transfection, seed 15,000 Human embryonic kidney 293A (HEK 293A) cells in 200 &#181;L of Dulbecco&#39;s ...

Take eukaryotic expression vectors, encoding either an antibody heavy chain or an antibody light chain, cloned from antigen-specific B cells.
Introduce polyethylenimine, a cationic polymer, that electrostatically interacts with the negatively-charged vectors, forming polyplexes.
Incubate mammalian cells with the polyplexes, which adhere to the cells and are internalized via endocytosis. The resulting endosomes fuse with lysosomes.
The polymers in the polyplexes cause osmotic swelling of the vesicle, leading to its rupture and release of the vectors.&#160;
The released vectors enter the nucleus and are transcribed into heavy-chain and light-chain mRNA, which undergo translation via ribosomes on the endoplasmic reticulum.
The resulting peptide chains assemble into antibodies, which are transported to the Golgi and packaged into secretory vesicles.
Upon fusion of the secretory vesicles and the cell membrane, the antibodies are released extracellularly.
Harvest the antibody-containing supernatant for downstream assays.

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

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