Rapid Isolation of Rare Isotype-Switched Hybridoma Variants: Application to the Generation of IgG2a and IgG2b MAb to CD63, a Late Endosome and Exosome Marker.

Stéphanie Charrin,Denis Clay,Guillaume Van Niel,Martine Billard,Claude Boucheix,Roberta Palmulli,Eric Rubinstein

doi:10.3390/antib9030029

Abstract

CD63, a member of the tetraspanin superfamily, is used as a marker of late endosomes and lysosome-related organelles, as well as a marker of exosomes. Here, we selected rare isotype variants of TS63 by sorting hybridoma cells on the basis of their high expression of surface immunoglobulins of the IgG2a and IgG2b subclass. Pure populations of cells secreting IgG2a and IgG2b variants of TS63 (referred to as TS63a and TS63b) were obtained using two rounds of cell sorting and one limited dilution cloning step. We validate that these new TS63 variants are suitable for co-labeling with mAb of the IgG1 subclass directed to other molecules, using anti mouse subclass antibodies, and for the labeling of exosomes through direct binding to protein A-coated gold particles. These mAbs will be useful to study the intracellular localization of various proteins and facilitate electron microscopy analysis of CD63 localization.

Highlights

CD63 is a member of the tetraspanin superfamily, proteins characterized by four transmembrane domains and a specific fold in the largest of the two extracellular domains [1,2]
To select cells expressing IgG2a or IgG2b at their cell surface, the hybridoma cells producing the TS63 mAb were incubated with Alexa 647-labelled goat anti-mouse IgG2a or IgG2b, or a Dylight
We were able to generate in a few weeks and with limited manipulations IgG2a and IgG2b variant of TS63, a mAb directed to CD63, a marker of late endosomes, lysosome-related organelles as well as exosomes

Summary

Introduction

CD63 is a member of the tetraspanin superfamily, proteins characterized by four transmembrane domains and a specific fold in the largest of the two extracellular domains [1,2]. CD63 plays a role in protein and membrane trafficking. It regulates the trafficking of other proteins such as the β-chain of the gastric H+/K+-ATPase, CXCR4, to intracellular compartments [3,4]. It regulates membrane trafficking, for example, the production of intra luminal vesicles in MVB, or IgE-mediated mast cell degranulation [5,6]. CD63 regulates HIV and papilloma virus infection [7,8]. Mice lacking CD63 have normal lysosomal functions but show a pigmentation defect due to alteration of melanosome biogenesis and a reduction of acute allergic reactions [6,9,10]

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Conclusion