Simultaneous quantitative analysis of multiple protein species within a single sample using standard scanning densitometry.

It is often desirable, when conducting Western blot analyses, to accurately quantify the relative expression of multiple target proteins in a single sample. A common problem occurs: however, when the target proteins vary beyond the linear range of the detection system; thus precluding accurate densitometric analysis for all samples. For example, analysis of teleost immunoglobulin structure under non-reducing but denaturing conditions, yields multiple, differentially polymerized forms (redox forms) within a single sample, which can exceed single log differences in concentration, as visualized by chemiluminescent and X-ray film development. To resolve this difficulty an efficient technique has been developed that uses dilutions of a single sample, allowing accurate quantification of target proteins within their potentially unique and varied linear range of detection. Upon consideration of the respective dilution factor that yields an appropriate estimate, the multiple targets can be quantified. When the results from this technique are compared to other systems possessing more expansive linear ranges, the results obtained are comparable to within 1%. Thus, laboratories without access to more sensitive and costly densitometric instrumentation can still employ standard densitometric analysis to accurately quantify multiple targets in a single sample.

The development of a real-time biosensor for the detection of trace levels of trinitrotoluene (TNT) in aquatic environments.

This paper describes the development of a highly sensitive TNT immunosensor consisting of a highly specific monoclonal antibody coupled with a prototype fluorescence-based detector system (KinExA Inline Biosensor, Sapidyne Instrument Inc). The antibody developed possesses a high affinity for TNT (association constant, aK 8.2) with minimal cross reactivity with other compounds such as tetryl, 2,4-dinitrotoluene and 2-amino-4,6-dinitrotoluene. This system provides sample assessment within 160 s from source acquisition and possesses sensitivity for TNT of 0.05 microg/L in ground water. The sensor can be regenerated in 8 min, allows a minimum of 40 repeated readings, and has a standard error of 0.1-0.4% between repeat readings. The fluidics and software allow samples to be obtained from up to eight different sources allowing the user to examine the stratification of the pollutant in the water column. We believe that this immunosensor can be used to rapidly assess trace levels of TNT in environmental water samples.

Infection of barramundi Lates calcarifer with Streptococcus iniae: effects of different routes of exposure.

The use of various challenge techniques has allowed the formation of a hypothesis for the mode of infection of Streptococcus iniae in barramundi. A bacterial dose of 1 x 10(3) colony forming units (cfu), corresponding to the LD50, delivered orally to barramundi could initiate the sub-acute form of the disease observed at the farms. The acute form of the disease could be initiated through bath exposure to the pathogen. S. iniae was equally as infective in freshwater, saltwater or when fish were subject to skin trauma prior to exposure, with LD50 values of 3.2 x 10(4), 2.0 x 10(4), 3.2 x 10(4) cfu, respectively, when observed over a 10 d period. It is suggested that sub-acute infection occurs orally, with mass mortalities occurring through the increased presence of the bacterium in the environment.

Streptococcus iniae, a bacterial infection in barramundi Lates calcarifer.

The cause of ongoing mortality in barramundi Lates calcarifer (Bloch) in seawater culture was identified as Streptococcus iniae by biochemical and physiological tests. This is the first published record of this bacterial species in Australia and the first confirmed report of S. iniae causing mortality in barramundi. The bacterium was highly pathogenic for barramundi when challenged by bath exposure. The pathogen was found to have a LD50 of 2.5 x 10(5) and 3.2 x 10(4) colony-forming units at 48 h and 10 d respectively. Experimental challenge of barramundi resulted in high levels of mortality (> 40%) within a 48 h period. Ten days after the challenge, S. iniae could not be isolated from kidney, spleen, liver or eye of surviving fish. However, the organism was easily isolated from the brain of both moribund and healthy fish, indicating that barramundi can carry the bacterium asymptomatically.