A hexamer peptide ligand that binds selectively to staphylococcal enterotoxin B: isolation from a solid phase combinatorial library.

By screening a solid-phase combinatorial peptide library, a short peptide ligand, YYWLHH, has been discovered that binds with high affinity and selectivity to staphylococcal enterotoxin B (SEB), but only weakly to other SEs that share sequence and structural homology with SEB. Using column affinity chromatography with an immobilized YYWLHH stationary phase, it was possible to separate SEB quantitatively from Staphylococcus aureus fermentation broth, a complex mixture of proteins, carbohydrates and other biomolecules. The immobilized peptide was also used to purify native SEB from a mixture containing denatured and hydrolyzed SEB, and showed little cross-reactivity with other SEs. To our knowledge this is the first report of a highly specific short peptide ligand for SEB. Such a ligand is a potential candidate to replace antibodies for detection, removal and purification strategies for SEB.

Gangliosides as receptors for biological toxins: development of sensitive fluoroimmunoassays using ganglioside-bearing liposomes.

Gangliosides, glycosphingolipids present in the membranes of neuronal and other cells, are natural receptors for a number of bacterial toxins and viruses whose sensitive detection is of interest in clinical medicine as well as in biological warfare or terrorism incidents. Liposomes containing gangliosides mimic cells that are invaded by bacterial toxins and can be used as sensitive probes for detecting these toxins. We discuss detection of three bacterial toxins-tetanus, botulinum, and cholera toxins using ganglioside-bearing liposomes. Tetanus and botulinum toxins selectively bind gangliosides of the G1b series, namely, GT1b, GD1b, and GQ1b; and cholera toxin binds GM1 very specifically. Unilamellar liposomes containing GT1b or GM1 as one of the constituent lipids were prepared by extrusion through polycarbonate membranes. To impart signal generation capability to these liposomes, fluorophore-labeled lipids were incorporated in the bilayer of liposomes. The fluorescent liposomes, containing both a marker (rhodamine) and a receptor (GT1b or GM1) in the bilayer, were used in sandwich fluoroimmunoassays for tetanus, botulinum, and cholera toxins and as low as 1 nM of each toxin could be detected. The apparent dissociation constants of liposome-toxin complexes were in 10(-8) M range, indicating strong binding. This is the first report on detection of tetanus and botulinum toxins based on specific recognition by gangliosides. The fluorescent liposomes are attractive as immunoreagents for another reason as well--they provide enormous signal amplification for each binding event as each liposome contains up to 22,000 rhodamine molecules. The present approach using receptors incorporated in bilayers of liposomes offers a unique solution to employ water-insoluble receptors, such as glycolipids and membrane proteins, for sensitive detection of toxins and other clinically important biomolecules.

Selective trace enrichment by immunoaffinity capillary electrochromatography on-line with capillary zone electrophoresis - laser-induced fluorescence.

Limited by the lack of a sensitive, universal detector, many capillary-based liquid-phase separation techniques might benefit from techniques that overcome modest concentration sensitivity by preconcentrating large injection volumes. The work presented employs selective solid-phase extraction by immunoaffinity capillary electrochromatography (IACEC) to enhance detection limits. A model analyte, fluorescein isothiocyanate (FITC) biotin, is electrokinetically applied to a capillary column packed with an immobilized anti-biotin-IgG support. After selective extraction by the immunoaffinity capillary, the bound analyte is eluted, migrates by capillary zone electrophoresis (CZE), and is detected by laser-induced fluorescence. The column is regenerated and reused many times. We evaluate the performance of IACEC for selective trace enrichment of analytes prior to CZE. The calibration curve for FITC-biotin bound versus application time is linear from 10 to 300 seconds. Recovery of FITC-biotin spiked into a diluted urinary metabolites solution was 89.4% versus spiked buffer, with a precision of 1.8% relative standard deviation (RSD).

Development of sensors for direct detection of organophosphates. Part I: Immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports.

Biosensors for organophosphates in solution may be constructed by monitoring the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (< 1 mm2) for microsensors. In order to construct microsensors with increased surface area for enzyme immobilization, we used a sol-gel process to create highly porous and stable silica matrices. Surface porosity of sol-gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this analysis, porous and non-porous silica beads were used as model substrates of sol-gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-gamma-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme activity. Enzyme was also immobilized to sol-gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.

Elliptical trough reflector for the collection of light from linear sources.

A trough reflector with a reflective, truncated elliptical surface was designed to efficiently collect freely propagating light from a linear source. The source was placed at one focus of the reflector, and light was collected through a rectangular aperture near the second focus. Collection efficiency was much greater than that of a spherical integrator and approximately 6.5x greater than that of an objective lens; as much as approximately 55% of the light could be captured from the full aperture. This reflector could be used to efficiently collect surface fluorescence excited by use of evanescent waves in fluorescence-based fiber optic or capillary waveguide sensors.

Hollow cylindrical waveguides for use as evanescent fluorescence-based sensors: effect of numerical aperture on collected signal.

Hollow cylindrical waveguide sensors permit conventional capillary injection techniques for flowing precise volumes of a liquid sample through the sensor while exciting and collecting fluorescence by use of evanescent fields. Both a ray-optics model and experimental data show that fluorescence collected from bulk or thin-layer fluorescent samples increases strongly as the numerical aperture (N.A.) increases and is maximized when the N.A. of the excitation-collection optics matches the waveguide N.A. The dependence of fluorescence on N.A. closely resembled that reported previously for solid cylindrical waveguides. Mode mixing reduced the strength of this dependence and should be minimized to increase collected fluorescence.

A modified imaging sequence for accurate T2 measurements using NMR microscopy.

A modified spin-echo pulse sequence is described that enables accurate T2 measurements to be made in NMR microimaging experiments. The modified sequence eliminates cumulative diffusion losses that lead to an underestimation of the T2 relaxation time using conventional spin-echo pulse sequences. The approach is theoretically justified and confirmed in comparative experiments on phantoms.

A modified quadrupole gradient set for use in high resolution MRI tagging.

A special purpose gradient set with a Cos(2 theta) current distribution has been constructed for high resolution MR imaging of small samples in a clinical Signa 1.5 T scanner using the existing gradient amplifiers. The X, Y, and Z gradient coils can attain gradient field strengths of 11.3, 4.7, and 15.2 G/cm at 100 amps current, respectively, with a slew rate of 20 G/cm/ms and usable ramp time of 150 microseconds. Field distortions are less than 2% over the central 8 cm of the bore, suitable for high resolution tagging of isolated canine hearts.

The design and construction of a NMR microscopy probe.

The design and construction of a probe for proton NMR microimaging studies of small objects, in particular single cells less than 1 mm in diameter, at 8.5 T is described. The probe incorporates a novel RF assembly and novel semishielded quadrupolar gradient coils. The design criteria are discussed and some preliminary results presented.

Relaxation-time and diffusion NMR microscopy of single neurons.

Relaxation-time and diffusion-weighted NMR micrographs have been obtained for single neurons isolated from Aplysia californica. These images allow the nucleus and cytoplasm to be clearly differentiated, in contrast to proton spin-density images, which appear relatively homogenous. Images of the spatial distribution of T1 and T2 relaxivities and the diffusion coefficient (D), as well as average values for T1, T2, and D in the cytoplasm and nucleus, were calculated from sets of appropriately weighted images. In all cases, water in the nucleus had relaxation and diffusion properties markedly differing from those of cytoplasmic water, which in turn had properties which were distinct from those of free water. Additionally, the cytoplasmic T2 was observed to triple following cell death, which is attributed to cytoplasmic dilution as water enters the cell. The work presented represents the first effort at a consistent exploration of the spatial distribution of NMR characteristics of water within intact single cells. These studies have implications both for modeling the NMR characteristics of water in neuronal tissues based on an understanding of the characteristics of water in different cell compartments and for understanding water/macromolecule interactions within cells. NMR microscopy studies such as these may help form a foundation for understanding and interpreting NMR characteristics measured from large assemblies of cells, i.e., spectroscopy and imaging of living tissues.

Gel-entrapment of perfluorocarbons: a fluorine-19 NMR spectroscopic method for monitoring oxygen concentration in cell perfusion systems.

Oxygenation is a major determinant of the physiological state of cultured cells. 19F NMR can be used to determine the oxygen concentration available to cells immobilized in a gel matrix by measuring the relaxation rate (1/T1) of perfluorocarbons (PFC) incorporated into the gel matrix. In calcium alginate gel beads without cells the relaxation rate (1/T1) of the trifluoromethyl group of perfluorotripropylamine (FTPA) varies linearly with oxygen concentration, with a slope of 1.26 +/- 0.15 x 10(-3) s-1 microM-1 and an intercept of 0.50 +/- 0.04 s-1. During perfusion with medium equilibrated with 95%/5% O2/CO2, changes in PFC T1s indicate that the average oxygen concentration was reduced from 894 +/- 102 microM in the absence of cells to 476 +/- 65 microM and 475 +/- 50 microM in the presence of 0.7 x 10(8) EMT6/Ro and RIF-1 murine tumor cells per milliliter of gel, respectively. The presence of 0.2 microliters of FTPA/ml of gel had no effect on the energy status of the cells as indicated by 31P NMR spectra. To calculate oxygen gradients within the beads from the average PFC T1 of the sample, a mathematical model was used assuming that oxygen is the limiting nutrient for cell metabolism and that the cellular oxygen consumption rate is independent of oxygen concentration. Data for EMT6/Ro cells were fit using experimentally determined perfusion parameters together with literature values for cell volume and oxygen consumption rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Fourier analysis of cell motility: correlation of motility with metastatic potential.

We report the development of a computerized, mathematical system for quantitating the various types of cell motility. This Fourier analysis method simultaneously quantifies for individual cells (i) temporal changes in cell shape represented by cell ruffling, undulation, and pseudopodal extension, (ii) cell translation, and (iii) average cell size and shape. This spatial-temporal Fourier analysis was tested on a series of well-characterized animal tumor cell lines of rat prostatic cancer to study in a quantitative manner the correlation of cell motility with increasing in vivo metastatic potential. Fourier motility coefficients measuring pseudopodal extension correlated best with metastatic potential in the cell lines studied. This study demonstrated that Fourier analysis provides quantitative measurement of cell motility that may be applied to the study of biological processes. This analysis should aid in the study of the motility of individual cells in various areas of cellular and tumor biology.