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J Immunol Methods. 2008 Apr 20;333(1-2):156-66. doi: 10.1016/j.jim.2008.01.015. Epub 2008 Feb 20.

Hybridoma populations enriched for affinity-matured human IgGs yield high-affinity antibodies specific for botulinum neurotoxins.

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1
Cardeza Foundation for Hematologic Research and Kimmel Cancer Center, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA 19107, United States.

Abstract

The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against infectious diseases and bioterror agents. Hybridoma methods for cloning these antibodies have many potential advantages, including convenience, high-yield antibody expression, and the ability to capture the antibodies in their native configurations. However, they have been hindered by hybridoma instability and limited accessibility of antigen-specific, class-switched human B-cells. Here, we describe an efficient, three-step method that uses human peripheral blood B-cells to produce stable hybridoma populations that are highly-enriched for affinity-matured human IgG antibodies. Peripheral blood mononuclear cells (PBMCs) are (a) selected for expression of CD27, a marker of post-germinal center B-cells, (b) cultured in vitro to promote B-cell proliferation and class-switching, and (c) fused to a genetically modified myeloma cell line. Using this strategy, we cloned 5 IgG antibodies that bind botulinum neurotoxins (BoNT), the causes of the food-borne paralytic illness, botulism, and Category A Select Bioterror agents. Two of these antibodies bind BoNT with low picomolar affinities. One (30B) is the first high-affinity human antibody to bind serotype B BoNT, and another (6A) is able to neutralize a lethal dose of serotype A BoNT in vivo in pre- and post-exposure models. This optimized hybridoma method will broadly enable access to the native human antibody repertoire.

PMID:
18313069
DOI:
10.1016/j.jim.2008.01.015
[Indexed for MEDLINE]
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