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Abstracts
Workgroup: Frontiers in Disease Research
Monoclonal antibody ELISA assay for detection of Perkinsus marinus in oyster tissues
Principal Investigator(s):
Christopher F. Dungan, Maryland Dept. of Natural Resources, Oxford Laboratory, Oxford, MD, cdungan@dnr.state.md.us
Funding Period: 9/1/98 - 1/31/01
Stable hybridomas secreting murine IgG1 monoclonal antibodies (MAB) to Perkinsus marinus epitopes were produced, cloned, cryopreserved, and archived under liquid nitrogen refrigeration at two locations
Ten murine IgG1 MABs were sub-isotyped, produced (5mg) by hybridoma culture, chromatographically purified, and their binding epitopes characterized on western blots of solubilized and electrophoretically-resolved P. marinus proteins. Five MABs bound to primary structure epitopes of single 57-59kD reduced proteins. One MAB labeled a primary structure epitope present in both 31kD and 28kD reduced proteins. One MAB (16C11) labeled a ubiquitous, recurrent, primary structure epitope on >13 reduced proteins of 14-66kD sizes. Three MABs labeled secondary structure epitopes on unreduced proteins, which were not labeled on reduced proteins in western blot assays.
Binding specificity testing with MAB immunoassays of histological sections of aquatic hosts infected by diverse parasites, including different Perkinsus species, were thwarted by the failure of 9/10 of our MABs to label P. marinus in histological sections from infected oysters. Like our prior rabbit polyclonal antibodies to P. marinus, MAB 16C11 specifically and intensely labeled formalin-fixed P. marinus and Perkinsus sp. cells in histological sections from infected oysters, clams, scallops, and abalone, did not label Haplosporidium nelsoni cells in oyster sections, but did label Hematodinium spp. parasitic dinoflagellates infecting three decapod crustacean species.
Due to our inability to validate mono-specificity for P. marinus in any of our MABs, diagnostic ELISA assay development was abandoned pending future production of MABs with more robust binding epitopes that are unique to P. marinus, or collection of a suite of infected tissue samples from diverse hosts and parasites, that are suitable to determining MAB binding specificities in non-histological assays.
IMPACTS and/or BENEFITS:
Results confirm the feasibility of producing MABs to P. marinus epitopes, using antigens extracted from axenic in vitro parasite isolate cultures as both immunogens and hybridoma screening assay antigens, and confirm the utility and shortcomings of several assay types for screening and characterization of such MABs. These results will inform future efforts.
If convenient histological specificity testing assays are included in future diagnostic MAB production efforts, formaldehyde treatment of immunogens, but not of hybridoma screening assay antigens, may result in selection for cloning of hybridomas secreting MABs that bind to epitopes present in both fixed (histological) and unfixed (host tissue homogenate) samples.
Benefits of MAB-based diagnostic immunoassays include the perennial availability of characterized, specific antibody reagents from immortal hybridoma cell lines. This MAB attribute reduces assay reagent costs, enables assay specification standardization and precision, and promotes the commercial production of rapid, low cost, high-sensitivity immunossay systems.
PROJECT PUBLICATIONS:
Dungan C.F. and Hamilton R.M. 2001. Production and binding specificities of MABs to Perkinsus marinus cellular antigens. J. Shellfish Res. 20:543 (abstract)
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