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Abstracts
Workgroup: Frontiers in Disease Research
Rabbit polyclonal antibody ELISA assay for detection of Perkinsus marinus infections 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: 4/1/94 - 12/31/96
Two polyclonal rabbit antisera to Perkinsus marinus cellular immunogens were produced, purified, specificity-tested, and labeled.
Using biotinylated and unconjugated preparations of the same rabbit IgG antibodies, we designed, optimized, and tested the clinical performance of an immuno-affinity, antigen-capture, microplate ELISA assay for quantitative detection of P. marinus antigens in heterogeneous oyster tissue homogenates.
The optimized ELISA assay format, which utilized a biotin/streptavidin-complex detection system, quantitatively detected as little as 10ng of P. marinus protein in oyster tissue homogenates (40:g of oyster protein), with assay absorbance signals proportional to parasite protein concentrations in spiked oyster tissue homogenates.
The optimized ELISA assay reliably and quantitatively detected P. marinus infections in clinical samples categorized to harbor light, moderate, and heavy infections by standard Ray's fluid thioglycollate medium (RFTM) assays, and ELISA assay signals were linearly correlated with categorical infection intensities estimated by paired RFTM solid tissue assays.
The optimized ELISA assay failed to detect P. marinus infections in 3% of oysters that were independently diagnosed as infected by RFTM assays of replicate tissue sub-samples (false-negative errors), but yielded positive diagnoses for 81% of oysters diagnosed as uninfected by RFTM assays. The latter differential reflects the increased sensitivity and accuracy of the ELISA assay over the standard RFTM assay.
IMPACTS and/or BENEFITS:
Results validated a newly developed diagnostic assay for rapid, sensitive, quantitative, and automated detection of P. marinus infections in oyster tissues.
Diagnostic results from ELISA are available in hours, compared to days for existing assays, and reduced analytical effort per analyzed sample may reduce diagnostic costs, concurrent with increasing the speed and accuracy of dermo disease diagnostic assays.
Assay antibodies have been liberally distributed to diverse oyster disease research investigations, and have also been distributed for commercial diagnostic assay development.
PROJECT PUBLICATIONS:
Dungan C.F. and Hamilton R.M. 1997. Microplate ELISA assay for detection of Perkinsus marinus in oyster tissues. J. Shellfish Res. 16:330-331 (abstract)
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