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J Vis Exp. 2018 Feb 6;(132). doi: 10.3791/56508.

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood.

Author information

1
Centre Régional de Recherche en Cancérologie et Immunologie Nantes/Angers, INSERM 1232, LabEx IGO, Université de Nantes; CHU Nantes.
2
Centre Régional de Recherche en Cancérologie et Immunologie Nantes/Angers, INSERM 1232, LabEx IGO, Université de Nantes; Xavier.Saulquin@univ-nantes.fr.
3
Centre Régional de Recherche en Cancérologie et Immunologie Nantes/Angers, INSERM 1232, LabEx IGO, Université de Nantes; Laetitia.Gautreau@univ-nantes.fr.

Abstract

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

PMID:
29443062
PMCID:
PMC5912359
[Available on 2020-02-06]
DOI:
10.3791/56508
[Indexed for MEDLINE]

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