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BMC Biology

, 8:76

First Online: 04 June 2010Received: 06 May 2010Accepted: 04 June 2010

Abstract

BackgroundMonoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas.

ResultsIncreased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos.

ConclusionsThis method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.

AbbreviationsBSAbovine serum albumin

CMVcytomegalovirus

IgGimmunoglobulin G

PBSphosphate buffered saline

PBSTPBS Tween

PCRpolymerase chain reaction

RT-PCRreverse transcription PCR.

Electronic supplementary materialThe online version of this article doi:10.1186-1741-7007-8-76 contains supplementary material, which is available to authorized users.

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Author: Cécile Crosnier - Nicole Staudt - Gavin J Wright

Source: https://link.springer.com/article/10.1186/1741-7007-8-76







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