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Vet Immunol Immunopathol. 2013 Jun 15;153(3-4):187-93. doi: 10.1016/j.vetimm.2013.02.010. Epub 2013 Feb 19.

Production of seven monoclonal equine immunoglobulins isotyped by multiplex analysis.

Author information

1
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

Abstract

Horses have 11 immunoglobulin isotypes: IgM, IgD, IgA, IgE, and seven IgG subclasses designated as IgG1-IgG7, each of which are distinguished by separate genes encoding the constant heavy chain regions. Immunoglobulin (Ig) isotypes have different functions during the immune response and pathogen-specific isotypes can be used as indicators for immunity and protection from disease. In addition to existing monoclonal antibodies to various equine Igs, quantification of the individual isotypes requires pure isotype standards. In this report, we describe a fusion between X63-Ag8.653 mouse myeloma cells and horse PBMC to create equine-murine heterohybridomas. Initial screening for Ig production was performed by ELISA. Further testing was performed by a new 5-plex fluorescent bead-based assay able to simultaneously detect equine IgM, IgG1, IgG4/7, IgG5, and IgG6. Production of IgG3 and IgE was tested by separate bead assays. Seven stable heterohybridoma clones producing monoclonal equine IgM, IgG1, IgG3, IgG4/7, IgG5, IgG6 and IgE were created. Purified Ig isotypes were then tested by SDS-PAGE. The pure, monoclonal equine Ig isotypes and the new equine Ig multiplex testing developed here are valuable tools to quantify antibody responses and to accurately determine individual isotypes concentrations in horses.

PMID:
23541920
DOI:
10.1016/j.vetimm.2013.02.010
[Indexed for MEDLINE]

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