Martin S. Piltch

A novel method for preclinical detection of PrPSc in blood.

In this study, we demonstrate that a moderate amount of protein misfolding cyclic amplification (PMCA) coupled to a novel surround optical fibre immunoassay (SOFIA) detection scheme can be used to detect the disease-associated form of the prion protein (PrP(Sc)) in protease-untreated plasma from preclinical and clinical scrapie sheep, and white-tailed deer with chronic wasting disease, following natural and experimental infection. PrP(Sc), resulting from a conformational change of the normal (cellular) form of prion protein (PrP(C)), is considered central to neuropathogenesis and serves as the only reliable molecular marker for prion disease diagnosis. While the highest levels of PrP(Sc) are present in the central nervous system, the development of a reasonable diagnostic assay requires the use of body fluids that characteristically contain exceedingly low levels of PrP(Sc). PrP(Sc) has been detected in the blood of sick animals by means of PMCA technology. However, repeated cycling over several days, which is necessary for PMCA of blood material, has been reported to result in decreased specificity (false positives). To generate an assay for PrP(Sc) in blood that is both highly sensitive and specific, we have utilized limited serial PMCA (sPMCA) with SOFIA. We did not find any enhancement of sPMCA with the addition of polyadenylic acid nor was it necessary to match the genotypes of the PrP(C) and PrP(Sc) sources for efficient amplification.

Prion Disease Detection, PMCA Kinetics, and IgG in Urine from Sheep Naturally/Experimentally Infected with Scrapie and Deer with Preclinical/Clinical Chronic Wasting Disease

ABSTRACT Prion diseases, also known as transmissible spongiform encephalopathies, are fatal neurodegenerative disorders. Low levels of infectious agent and limited, infrequent success of disease transmissibility and PrPSc detection have been reported with urine from experimentally infected clinical cervids and rodents. We report the detection of prion disease-associated seeding activity (PASA) in urine from naturally and orally infected sheep with clinical scrapie agent and orally infected preclinical and infected white-tailed deer with clinical chronic wasting disease (CWD). This is the first report on PASA detection of PrPSc from the urine of naturally or preclinical prion-diseased ovine or cervids. Detection was achieved by using the surround optical fiber immunoassay (SOFIA) to measure the products of limited serial protein misfolding cyclic amplification (sPMCA). Conversion of PrPC to PrPSc was not influenced by the presence of poly(A) during sPMCA or by the homogeneity of the PrP genotypes between the PrPC source and urine donor animals. Analysis of the sPMCA-SOFIA data resembled a linear, rather than an exponential, course. Compared to uninfected animals, there was a 2- to 4-log increase of proteinase K-sensitive, light chain immunoglobulin G (IgG) fragments in scrapie-infected sheep but not in infected CWD-infected deer. The higher-than-normal range of IgG levels found in the naturally and experimentally infected clinical scrapie-infected sheep were independent of their genotypes. Although analysis of urine samples throughout the course of infection would be necessary to determine the usefulness of altered IgG levels as a disease biomarker, detection of PrPSc from PASA in urine points to its potential value for antemortem diagnosis of prion diseases.

Surround optical fiber immunoassay (SOFIA): an ultra-sensitive assay for prion protein detection.

We describe the development of a new technology (SOFIA) and demonstrate its utility by establishing a sensitive and specific assay for PrP(Sc). SOFIA is a surround optical fiber immunoassay which is comprised of a set of specific monoclonal antibodies and comprehensive capture of high energy fluorescence emission. In its current format, this system is capable of detecting less than 10 attogram (ag) of hamster, sheep and deer recombinant PrP. Approximately 10 ag of PrP(Sc) from 263 K-infected hamster brains can be detected with similar lower limits of PrP(Sc) detection from the brains of scrapie-infected sheep and deer infected with chronic wasting disease. These detection limits allow protease treated and untreated material to be diluted beyond the point where PrP(C), non-specific proteins or other extraneous material may interfere with PrP(Sc) signal detection and/or specificity. This not only eliminates the issue of specificity of PrP(Sc) detection but also increases sensitivity since the possibility of partial PrP(Sc) proteolysis is no longer a concern. SOFIA will likely lead to early antemortem detection of transmissible encephalopathies and is also amenable for use with additional target amplification protocols. SOFIA represents a sensitive means for detecting specific proteins involved in disease pathogenesis and/or diagnosis that extends beyond the scope of the transmissible spongiform encephalopathies.

Multi-line pulsed CO2 oscillator

Abstract For use in laser-induced fusion investigations, a CO2 laser oscillator was developed whose output spectrum contains three simultaneously appearing lines of equal intensity. An etalon filter provided the required wavelength dependent loss.

Temperature-dependent Sellmeier coefficients and nonlinear optics average power limit for germanium

A temperature-dependent Sellmeier equation is calculated for Ge. The accuracy of this temperature-dependent Sellmeier equation is compared with the internal consistency of the data. The phase-matching condition is discussed for four-wave mixing when two of the wave vectors are equal. The average power limit for four-wave mixing in a material like Ge is derived both for the case where one wave vector is fixed and for the case where two wave vectors are fixed. Using the derived Sellmeier equation, the average power limit for the four-wave mixing is calulated.

Pulsed infrared difference frequency generation in CdGeAs2

The disclosure relates to a laser apparatus for generating a line-tunable pulsed infrared difference frequency output. The apparatus comprises a CO2 laser which produces a first frequency, a CO laser which produces a second frequency and a mixer for combining the output of the CO2 and CO lasers so as to produce a final output comprising a difference frequency from the first and second frequency outputs.

Temperature-dependent Sellmeier coefficients and coherence length for cadmium telluride*

A temperature-dependent Sellmeier equation is calculated for CdTe. Using the derived Sellmeier equation, the coherence length for second harmonic generation was calculated as well as the variation of the coherence length with temperature. The predicted coherence length is found to be in good agreement with measured values.

CF(4) laser oscillator-amplifier measurements: small signal gain and self-absorption.

Some of the fundamental parameters associated with the CF4 optically pumped laser were measured with the aid of an oscillator–amplifier arrangement. The small signal gain for the strongest P-branch line was determined for pure CF4 and for CF4 in the presence of He and CO buffer gases at different pressures. The self-absorption of the laser radiation by CF4 was determined for various P-, Q-, and R-branch lines at several temperatures. The data were used to determine that the loss mechanism for the laser process is associated with hot-band absorption from the 1 ν2 level.

Surface-enhanced Raman scattering in insulating materials by artificial plasmon production: Application to uranium compounds

The concept of surface-enhanced Raman scattering (SERS) is reviewed. The currently held position within the spectroscopic community is that the enormous SERS enhancement effect, sometimes as much as fourteen orders of magnitude, can be partially related to surface roughness but mostly to surface plasmons (polaritons). The plasmon states are absent in electrical insulators, thus precluding the applicability of the powerful SERS technique in analysing the surface conditions of these materials. This communication describes a technique for extending the SERS process to materials that have little or no intrinsic electrical conductivity.

Optical Pumping Of The v3 + v4 Band Of The CF 4 Molecule

The possible development of a room-temperature, 16 μm CF4 laser optically pumped by 5 μm radiation from a frequency-doubled CO, laser has been iyvestigated. This laser would utilize pumping ofithe v3 + v4 band of the CF4 at 1916-1 cm with subsequent lasing to v3 levels at 1283 cm-1 where there is essentially no thermally excited population. A spectrophone instrument was used to measure absorption cross sections of CF4 at the second harmonic frequencies of 22 of the 10 µm CO2 laser transitions. Lasing experiments to detect transitions at 16 μm are described.