Biosensor applications in contaminated estuaries: Implications for disaster research response.

BACKGROUND: Given the time and monetary costs associated with traditional analytical chemistry, there remains a need to rapidly characterize environmental samples for priority analysis, especially within disaster research response (DR2). As PAHs are both ubiquitous and occur as complex mixtures at many National Priority List sites, these compounds are of interest for post-disaster exposures. OBJECTIVE: This study tests the field application of the KinExA Inline Biosensor in Galveston Bay and the Houston Ship Channel (GB/HSC) and in the Elizabeth River, characterizing the PAH profiles of these region's soils and sediments. To our knowledge, this is the first application of the biosensor to include soils. METHODS: The biosensor enables calculation of total free PAHs in porewater (C free), which is confirmed through gas chromatography-mass spectrometry (GC-MS) analysis. To determine potential risk of the collected soils the United States Environmental Protection (USEPA) Agency's Regional Screening Level (RSL) Calculator is used along with the USEPA Region 4 Ecological Screening Values (R4-ESV) and Refined Screening Values (R4-RSV). RESULTS: Based on GC-MS results, all samples had PAH-related hazard indices below 1, indicating low noncarcinogenic risks, but some samples exceeded screening levels for PAH-associated cancer risks. Combining biosensor-based C free with Total Organic Carbon yields predictions highly correlated (r > 0.5) both with total PAH concentrations as well as with hazard indices and cancer risks. Additionally, several individual parent PAH concentrations in both the GB/HSC and Elizabeth River sediments exceeded the R4- ESV and R4-RSV values, indicating a need for follow-up sediment studies. CONCLUSIONS: The resulting data support the utility of the biosensor for future DR2 efforts to characterize PAH contamination, enabling preliminary PAH exposure risk screening to aid in prioritization of environmental sample analysis.

IgM-bearing B cell affinity subpopulations possess differential antigen sensitivity in rainbow trout.

The need for accurate assessments of in vitro generated antibody prompted examination of the effect of antigen on secreted antibody concentrations and affinities. It was found that the antigen concentrations commonly employed for in vitro stimulation were able to significantly compromise IgM titer and affinity estimates in rainbow trout. Specifically, IgM titers were underestimated with the high affinity antibodies being specifically blocked. To remedy this, pulsed antigen cultures were employed, and it was found to reveal more accurate IgM antibody titers and affinity estimates. Additionally, pulsed dose responses provided evidence that high antigen concentrations specifically suppressed high affinity B cell induction. Optimal expression of high affinity antibodies required exposure to lower concentrations of antigen. Each affinity subpopulation appeared to possess a graded sensitivity to each dose of antigen, revealing the complex dynamic for differential IgM-bearing B cell induction that is possible during a response. These results reveal not only the need for antigen removal prior to in vitro antibody secretion, but also the possible role of high zone immunological tolerance on IgM affinity maturation in rainbow trout.

Production and characterization of a mouse monoclonal antibody against smallmouth bass (Micropterus dolomieu) IgM.

A murine monoclonal antibody (mAb, IgG2a) was produced for the detection of smallmouth bass (Micropterus dolomieu) immunoglobulin (IgM). The antibody is specific for IgM heavy chain and was shown to also recognize the Ig heavy chain of largemouth bass (Micropterus salmoides) and bluegill (Lepomis macrochirus) using Western Blot analysis of plasma from 9 teleost taxa. When applied to the analysis of smallmouth bass total plasma IgM using ELISA, the mAb was found to be effective when used in an inhibition kinetic assay.

Production of a monoclonal antibody against of muskellunge (Esox masquinongy) IgM heavy chain and its use in development of an indirect ELISA for titrating circulating antibodies against VHSV-IVB.

This study reports the development of a monoclonal antibody (designated 3B10) against the muskellunge (Esox masquinongy) IgM. The 3B10 monoclonal antibody (mAb) belongs to the IgG3 kappa isotype. Western blotting demonstrated that 3B10 mAb reacted primarily to muskellunge IgM heavy chain. 3B10 also reacted strongly with the IgM heavy chain of other esocids, including the northern pike (Esox lucius), tiger muskellunge (E. masquinongy x E. lucius), and, to a much lesser extent, the chain pickerel (E. niger). The 3B10 mAb did not bind to IgM from 10 other fish species resident in the Great Lakes basin. Using the 3B10 mAb, it was possible to determine the muskellunge Ig ability to bind to antigens. Using trinitrophenyl hapten conjugated to keyhole limpet hemocyanin (TNP-KLH) as the eliciting antigen, muskellunge Ig subclasses exhibited a range of affinities with log aK values 5.56-6.25 that is considered intermediate compared to other fish species. 3B10 mAb was used to develop and evaluate an indirect ELISA for the detection and quantitation of circulating antibodies against the viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb). Using the newly optimized assay, anti-VHSV-IVb antibodies were detected in sera of VHSV-IVb vaccinated muskellunge as well as from those of wild muskellunge sampled from an endemic waterbody. In addition to its use in immunoassays, the developed 3B10 mAb will enable future investigation aiming at deciphering immune mechanism of this important fish species to pathogens.

Evaluation of a time efficient immunization strategy for anti-PAH antibody development.

The development of monoclonal antibodies (mAb) with affinity to small molecules can be a time-consuming process. To evaluate shortening the time for mAb production, we examined mouse antisera at different time points post-immunization to measure titer and to evaluate the affinity to the immunogen PBA (pyrene butyric acid). Fusions were also conducted temporally to evaluate antibody production success at various time periods. We produced anti-PBA antibodies 7 weeks post-immunization and selected for anti-PAH reactivity during the hybridoma screening process. Moreover, there were no obvious sensitivity differences relative to antibodies screened from a more traditional 18-week schedule. Our results demonstrate a more time efficient immunization strategy for anti-PAH antibody development that may be applied to other small molecules.

Supplementation of Perkinsus marinus cultures with host plasma or tissue homogenate enhances their infectivity.

The protozoan oyster parasite Perkinsus marinus can be cultured in vitro in a variety of media; however, this has been associated with a rapid attenuation of infectivity. Supplementation of defined media with products of P. marinus-susceptible (Crassostrea virginica) and -tolerant (Crassostrea gigas, Crassostrea ariakensis) oysters alters proliferation and protease expression profiles and induces differentiation into morphological forms typically seen in vivo. It was not known if attenuation could be reversed by host extract supplementation. To investigate correlations among these changes as well as their association with infectivity, the effects of medium supplementation with tissue homogenates from both susceptible and tolerant oyster species were examined. The supplements markedly altered both cell size and proliferation, regardless of species; however, upregulation of low-molecular-weight protease expression was most prominent with susceptible oysters extracts. Increased infectivity occurred with the use of oyster product-supplemented media, but it was not consistently associated with changes in cell size, cell morphology, or protease secretion and was not related to the susceptibility of the oyster species used as the supplement source.