A novel sensitive and selective bioassay for human type I interferons.

We describe a novel MxA gene-induction assay for type I interferons (IFN-alpha and IFN-beta) based on the specific induction of the MxA gene in cultured human cells. Accumulated intracellular MxA protein is determined by immunologic measurement by a rapid method using commercially available materials. IFN activity can be measured accurately over a concentration range of 0.1-30 IU/ml. In contrast, type II IFN and other cytokines are not significantly detected. The MxA-induction assay has advantages in terms of specificity, reliability, and sensitivity over other methods for assaying type I IFN. It has also been adapted and validated for measuring the titers of anti-IFN-beta neutralizing antibodies in human sera.

A novel bioassay for the determination of neutralizing antibodies to IFN-beta1b.

We have adapted the new MxA gene-induction bioassay to measure neutralizing antibodies to interferon-beta1b (IFN-beta1b, the active ingredient in Betaseron) in sera from patients treated with Betaseron. This antibody assay has been validated to quantify neutralizing titers of 1:20 and above, with a precision of +/- 0.20 in log10. We have used this MxA gene-induction antibody assay to reinvestigate serum samples from multiple sclerosis (MS) patients treated with Betaseron. The titers measured were closely comparable to those obtained in antiviral assays. Data obtained by both methods show that neutralizing antibodies may appear and subsequently disappear over time in the sera of some patients treated with Betaseron. Sera from some patients contain binding antibodies to IFN-beta1b. It was shown that binding antibody titers do not correlate quantitatively or qualitatively with neutralizing antibody titers, and indeed, a number of patients develop high levels of binding antibodies but never form measurable levels of neutralizing antibodies.

Local depletion of monoamines induced with in vivo voltammetry in the cat brain.

A significant depletion of the electroactive monoamines and their metabolites in the vicinity of a carbon fiber microelectrode may be induced by in vivo staircase voltammetry in the brain, even if the duration of the voltammetric scans is relatively short (approximately 5 s). The variation of this depletion was determined in the extracellular fluid of the cat thalamus at different durations of the pauses separating consecutive measurements. Pauses not shorter than 5 min ensured a nearly full relaxation, so that at the beginning of each subsequent scan a virtually undisturbed environment surrounded the electrode. With pauses shorter than 5 min, it is still possible to monitor major changes in the monoamine concentration. Staircase scans separated with 45 s pauses, for example, were suitable to study the increase in monoamine levels after administration of reserpine, and release phenomena stimulated with KCl were monitored with frequently repeated voltammetric pulses. The electrochemically induced depletion, on the other hand, can be used for characterizing the dynamics of mass transport in the studied brain structure. This was demonstrated with staircase voltammetry alternated with pauses of 1-100 s, and with quasi-chronoamperometry. In vivo brain voltammetry is generally used for monitoring extracellular monoamine (including dopamine) levels. These may be significantly altered by the voltammetric measurement itself through depletion in the vicinity of the electrode. This effect can be minimized with appropriate selection of sampling intervals and other parameters of staircase voltammetry. Conversely, depletion and the following relaxation can be used for determining dynamic characteristics of the studied brain structure which would be difficult to obtain otherwise.

Electrochemical calibration of in vivo brain dialysis samplers.

Measurement of state-related changes in the concentration of an endogenous substance using the in vivo dialysis technique requires a definition of the factors which relate the concentration detected in the outflowing perfusate to the concentration actually occurring in the extracellular space in which the sampler is located. In determine these factors, a rapid and highly accurate detector system is required which measures the concentration recovery in the perfusate and the dead space of the entire sampling system. To reduce the limitations of the microdialysis technique, two electrochemical microcells were developed for calibration of dialysis probes by computerized voltammetry. The electrochemically calibrated samplers were implanted into freely moving cats to measure concentration changes of monoamine metabolites in synchrony with sleep stages identified by polygraphy in order to demonstrate the applicability of electrochemical calibration in dialysis methods used in behavioral investigations.

Application of multidimensional affinity high-performance liquid chromatography and electrospray ionization liquid chromatography-mass spectrometry to the characterization of glycosylation in single-chain plasminogen activator. Initial results.

Preliminary results are presented using a combination of affinity chromatography, reversed-phase HPLC and electrospray ionization mass spectrometry to produced peptide maps for N-linked, O-linked and non-glycosylated peptides from an endoproteinase LysC digest of DSPA alpha 1, a recombinant DNA derived glycoprotein. Although the system was used to identify a number of major N-linked structures, notably complex biantennary structures attached to asparagine 362, no O-linked glycopeptides from the possible 4 attachment sites were identified. The system did, however, demonstrate the feasibility of the approach and the applicability of the instrumental system.

Application of capillary electrophoresis, high-performance liquid chromatography, on-line electrospray mass spectrometry and matrix-assisted laser desorption ionization--time of flight mass spectrometry to the characterization of single-chain plasminogen activator.

The analysis of recombinant Desmodus salivary plasminogen activator (DSPA alpha 1), a heterogeneous glycoprotein, is demonstrated through the use of high-performance liquid chromatography (HPLC), high-performance capillary electrophoresis (HPCE), liquid chromatography-electrospray mass spectrometry (LC--ES-MS), and matrix-assisted laser desorption ionization--time of flight mass spectrometry (MALDI--TOF-MS). The proteins is analyzed at three specific levels of detail: the intact protein, proteolytic digests of the protein, and fractions from the proteolytic digest. A method for "on-column" collection of HPLC fractions for subsequent transfer and analysis by HPCE and MALDI--TOF-MS is shown.

Application of high-performance liquid chromatograph-electrospray ionization mass spectrometry and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry in combination with selective enzymatic modifications in the characterization of glycosylation patterns in single-chain plasminogen activator.

The application of high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus salivary plasminogen activator, a heterogeneous glycoprotein. The sample was initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carbohydrates or a combination of neuraminidase and O-glycosidase to remove sialic acid and O-linked carbohydrates. By comparison of the LC-ESI-MS peptide maps for the fully glycosylated and deglycosylated samples, it was possible to unambiguously identify the sites of N-linked glycosylation as well a number of N-linked glycopeptides. The O-link glycopeptides, which are present at low level ( < 1%), were not detected prior to the deglycosylation, nor could changes in peptide elution in the map following deglycosylation be correlated with potential O-linked glycosylation sites.

Capillary zone electrophoresis of a recombinant adenovirus.

Adenovirus preparations are used as vectors in a number of gene therapy clinical development programs. The success of commercial production of adenovirus will strongly depend on the development of methods to define the recombinant virus product by analysis as opposed to being defined by the manufacturing process. While most analytical techniques examine portions of the virus, e.g. proteins or DNA, ion-exchange chromatography has been used to separate intact virus at low efficiency. A free zone capillary electrophoretic method was developed for high-efficiency separations of adenovirus 5. Experimental conditions such as buffer pH and concentration were explored which produced a high-efficiency separation in less than 20 min. The virus band was identified by collection of CE fractions and examination using a cell based assay. Initially, a single virus peak is found in fresh virus samples. After as little as one freeze-thaw in 1 x phosphate-buffered saline with 2% sucrose, the active virus migrates as a regular series of peaks. The nature of the virus modification leading to the differing electrophoretic mobilities is presently under investigation.

How to understand the response mechanism of ion-selective electrodes.

The present study breaks with the earlier mechanism of electrode potential on basis of experimental investigations and theoretical considerations. It rejects that the transport through the membrane produces the electrode potential and definitely proves that the electrode potential is created via surface chemisorption; i.e., the electrode potential is produced by a surface reaction. The reaction centres can be acid-base groups or complex formation groups (e.g., valinomycin or other alkaline earth metal complexing ligands.

Properties and analytical applications of the heteropolymolybdates of phosphorus, arsenic, silicon and germanium-I Spectrophotometric examination of the heteropoly acids.

Spectrophotometric studies have been made of the heteropoly acids of phosphorus, arsenic, silicon and germanium, formed at molybdate concentrations of 10(-1)-10(-3)M. It was found that the heteropoly anion is quantitatively produced at a Z-value of 1.60 in the case of a quinquevalent central atom and 1.50 for a quadrivalent central atom, if the amount of the latter is not higher than 50% of the stoichiometric (Z is the number of moles of protons taken up by one mole of molybdenum.) On addition of further acid the anion takes up protons, but its absorbance remains unchanged if measured at a wavelength at which the isopolymolybdates show no absorption. At Z-values higher than 2.2-2.3, i.e., in highly acidic media, a decomposition reaction starts; its products are octamolybdate and molybdenyl ions. The different molar absorptivities and other "irregularities" found in the various analytical procedures in the literature are due not to the heteropoly acids but to the isopolymolybdate formed together with the former.

Properties and analytical applications of the heteropolymolybdates of phosphorus, arsenic, silicon and germanium-II Modifications of the heteropoly acids.

Phospho-, arseno-, silico- and germanomolybdic acids may all exist in an alpha- and a beta-modification. alpha-Silicomolybdic acid is formed if the degree of acidification is below [H(+)]/[Mo] = 1.5. At higher acid concentrations the beta-modification is formed, and spontaneously transformed into the alpha-form. Germanomolybdic acid behaves similarly, but the transformation is much faster. Arsenomolybdic acid is always formed as the alpha-modification and is transformed into the beta-modification only later and in a very slow reaction if the pH of the solution is below 1. Formation of the two modifications is also dependent on acid concentration in the case of phosphomolybdic acid, but both modifications are stable and no spontaneous transformation occurs. Compounds similar to the beta-modifications but showing different molar absorptivities are formed in acetone-containing solution.

End effects in flow-analysis and process systems.

The "end effects" in flow-analysis and process systems are investigated theoretically on the basis of the axially-dispersed plug-flow model. Three-section systems consisting of reactor, fore- or after-section and additional section are used. In the additional section back-mix flow is assumed to exist. The corresponding Peclet number has been found to be 0.01. The difference between the system responses to a delta-function input signal for finite and infinite length of the fore- or after-section is studied. Both the statistical moments of the system responses and the actual response curves are used for that purpose. The results can be used to evaluate the length of the fore- and after-sections needed in real flow-analysis and process systems in order that the phenomena taking place downstream or upstream of these two sections, respectively, will not play an important role in the system response.

Correlation between flame geometry and the absorbance of some elements in the presence of organic solvents.

The correlation between the changes in flame geometry and the sensitivity of atomic-absorption determination of silver, copper, iron and lead in the presence of various solvents (water, ethanol, n-propanol, methyl acetate, ethyl acetate and acetone) has been investigated. The flame geometry was studied by the Schlieren technique. Organic solvents, allowing a greater rate of introduction of solution, enable a greater sensitivity to be achieved. However, owing to the expansion of the reaction zone, the increase in sensitivity is not as great as expected from the amount of sample introduced into the flame.

Voltammetric determination of the chlorine content of aqueous solutions, using a silicone-rubber based graphite electrode.

A method for the determination of chlorine and hypochlorous acid in 0.1N sulphuric acid by voltammetry is described. The method may be applied to stirred and flowing solutions and has a possible use in the monitoring of sewage effluent systems.

Properties and analytical applications of the heteropolymolybdates of phosphorus, arsenic, silicon and germanium-III Examination of two- and three-component systems without separation.

By taking advantage of the possible variations in acid and molybdenum concentration, wavelength of measurement, alpha- and beta-modifications, and media, P, As, Si and Ge can be determined spectrophotometrically as the heteropolymolybdates without separation. Rapid procedures are proposed for the analysis of two- and three-component mixtures of these elements.

Properties and analytical applications of the heteropolymolybdates of phosphorus, arsenic, silicon and germanium-IV Determination of phosphomolybdic and silicomolybdic acids via the molybdenum content, with phenylfluorone.

Procedures are proposed for the determination of phosphorus and silicon in the ppM range, by extraction of phosphomolybdic and silicomolybdic acids with organic solvent, decomposition of the complex and spectrophotometric determination of its molybdenum content.

Selective ion-exchanger behaviour of neutral carrier ion-selective electrode membranes.

Radiotracer experiments confirm that neutral carrier ion-selective electrodes made from plasticized PVC are low-capacity selective ion-exchangers. The ion-exchange selectivity is closely correlated to potentiometric selectivity.

Atomic-absorption spectrometry of laser-nebulized samples.

Thermal nebulization of nickel-base alloys, corundum plates and residues of solutions was performed with a laser beam. The solid aerosol was aspirated into an acetylene-air flame by an ejector. Linear analytical curves in wide concentration ranges were established and an average relative standard deviation of 10% was found. The detection limit is in the ng range for iron and copper, and lower for zinc, cadmium and sodium.

Graphite furnace and flame atomic-absorption technique for thermoanalytical investigations.

Aerosol particles formed from the vapour of electrothermally heated substances were introduced into an acetylene-air flame for atomization and detection. Thus individual observations could be made on condensed phase processes taking place in the furnace. Curves of absorbance vs. furnace temperature for several zinc compounds were recorded and compared with the corresponding thermoanalytical DTG-curves.

Study of the potential response of solid-state chloride electrodes at low concentration ranges.

Ion-selective electrodes based on silver chloride precipitates have been investigated in the low concentration range, by use of a specially designed cell of small volume. Electrode potential measurements and silver determinations in the corresponding solutions by atomic-absorption spectrometry were made. The results prove that the potential response of these ion-selective electrodes in the low concentration ranges is governed by inequality of the ion concentrations in the boundary zone of the test solution contacting the electrode membrane. This is a result of adsorption-desorption processes, a dissolution process followed by recrystallization of the silver chloride at the electrode membrane surface, and photoreduction of silver ions at the electrode surface.