Methodological approach

Capture of Single Antigen-Specific B cells to Produce Human Monoclonal Antibodies to Pathogens

 

To achieve our research goals, we are making use of a very efficient method to clone and express immunoglobulins from human single B cells specific to viral antigens.

 

Briefly, peripheral B cells are isolated from PBMCs of infected individuals by Ficoll separation and magnetic B-cell enrichment. Single antigen-specific B cells are identified by staining with the biotinylated antigen, and fluorescently labeled antibodies to B-cell surface markers. Single B cells are then sorted into 96-well PCR plates using a single-cell FACS sorter.

 

Single-cell cDNA synthesis is performed, and followed by two successive rounds of PCR amplifications of the cDNA fragments encoding the immunoglobulin heavy-chain (IgH) and light chains (Igk and Igl). All PCR products are sequenced to perform detailed Ig gene sequence analyses.

 

Restriction sites are introduced by the nested primers during the second PCR, and are used to clone the IgH, Igk and Igl DNA fragments into human Ig-expressing vectors.

 

Monoclonal antibodies are produced by co-transfection of HEK-293T cells with IgH and IgL expression-vectors using the polyethylenimine-precipitation method.

 

The antibodies are affinity purified from culture supernatants, and are finally characterized in details at a molecular and functional level.

 
 

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Technical References

 

Cloning and expression of murine Ig genes from single B cells.

Tiller T, Busse CE, Wardemann H.

J Immunol Methods. 2009 Oct 31;350(1-2):183-93. doi: 10.1016/j.jim.2009.08.009. Epub 2009 Aug 27.

PMID: 19716372 [PubMed - indexed for MEDLINE]

 

A method for identification of HIV gp140 binding memory B cells in human blood.

Scheid JF, Mouquet H, Feldhahn N, Walker BD, Pereyra F, Cutrell E, Seaman MS, Mascola JR, Wyatt RT, Wardemann H, Nussenzweig MC.

J Immunol Methods. 2009 Apr 15;343(2):65-7. doi: 10.1016/j.jim.2008.11.012. Epub 2008 Dec 25.

PMID: 19100741[PubMed - indexed for MEDLINE] 

 

Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen.

Smith K, Garman L, Wrammert J, Zheng NY, Capra JD, Ahmed R, Wilson PC.

Nat Protoc. 2009;4(3):372-84. doi: 10.1038/nprot.2009.3.

PMID:19247287 [PubMed - indexed for MEDLINE] 

 

Expression cloning of human B cell immunoglobulins.

Wardemann H, Kofer J.

Methods Mol Biol. 2013;971:93-111. doi: 10.1007/978-1-62703-269-8_5.

PMID: 23296959 [PubMed - indexed for MEDLINE]

 

Updated on 12/05/2014

Funding Supports

 

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Thank you to all our sponsors !

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