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(P539) Lipovitellin quantification in sediment-dwelling copepods: a potential screening tool for endocrine disrupting chemicals.
Volz, David*,1, Cary, Tawnya1, Block, David1, Chandler, G. Thomas1, 1 University of South Carolina, Columbia, SC, USA
ABSTRACT- A cost-effective, high-throughput, and simple enzyme-linked immunosorbant assay (ELISA) was developed for copepod (Amphiascus tenuiremis) lipovitellin quantification in a 96-well plate format using a fluorescence microplate reader. Four different indirect competitive ELISA approaches were developed and tested for sensitivity and standard variability. When testing several secondary antibody conjugates and/or alternate substrates, we found that maximum sensitivity (≥150 pg lipovitellin) and reliability (R2>0.99) was approached by simply increasing the primary and secondary antibody incubation times. This assay involves competing reactions between free lipovitellin and cross-reactive polyclonal anti-amphipod (Leptocheirus plumulosus) vitellin IgG. Antibodies are detected by alkaline phosphatase-labeled secondary antibodies and 4-methylumbelliferyl phosphate (4-MUP) fluorescent substrates. This lipovitellin ELISA yields a dynamic standard curve (2-500 ng/mL) and low coefficients of variation (mean ± SD = 16.93 ± 9.72 %). We have reliably and accurately detected lipovitellin in as few as 4 copepods. Baseline lipovitellin titers were quantified in A. tenuiremis gravid females and ranged from 5-7 ng lipovitellin/gravid female. Lipovitellin quantification in copepods is a sensitive and useful endpoint when linked with reproductive and population-level endpoints measured in our life-cycle microplate screening assays. Male/female reproductive failure or feminizing/masculinizing effects such as lipovitellin suppression or induction can be conducted given this assay's sensitivity.
Key words: lipovitellin, ELISA, copepod, EDCs
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