A simple way to increase recovery of the expressed VH and VL genes in single-sorted human B cells

Cloning VH and VL genes from individual antigen-specific B cells is an attractive approach for producing monoclonal antibodies of the desired specificity. Current RT-PCR protocols, however, result in the successful identification of VH and VL gene pairs in about half of the sorted cells. Here, we de...

Full description

Bibliographic Details
Published in:BioTechniques
Main Authors: Sergey V Guselnikov, Tatyana N Belovezhets, Sergey V Kulemzin, Andrey A Gorchakov, Alexander V Taranin
Format: Article
Language:English
Published: Taylor & Francis Group 2019-10-01
Subjects:
B lymphocytes
heavy chain
immunoglobulin
light chain
single-cell PCR
Online Access:https://www.future-science.com/doi/10.2144/btn-2019-0079
Description
Summary:Cloning VH and VL genes from individual antigen-specific B cells is an attractive approach for producing monoclonal antibodies of the desired specificity. Current RT-PCR protocols, however, result in the successful identification of VH and VL gene pairs in about half of the sorted cells. Here, we demonstrate that single-cell RT-PCR is likely affected by stochastic factors, and that running PCRs in triplicate results in successful amplification of the expressed VH and VL genes in 90–100% of single sorted human B cells.
ISSN:0736-6205
1940-9818

Similar Items