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Spoilage fungi represent a major cause of food and economic losses and certain species, which may produce mycotoxins, may also pose a threat to human and animal health. Thus, food safety and quality management relies notably on a good knowledge of the involved species. This knowledge is notably based on their fast and reliable identification and on the study of abiotic factors affecting their growth such as food preservatives, which are commonly used in the food industry. In this context, the objectives of this PhD. thesis were to develop MALDI-TOF mass spectrometry for mold identification and to evaluate its potential for species complex differentiation and strain typing, and finally, to evaluate the use of laser nephelometry to monitor fungal growth in the presence of food preservatives.First, a robust database was developed with 6500 spectra corresponding to 136 spoilage fungi. Then, MALDI-TOF MS was successfully applied to differentiate 23 species of Aspergillus section Flavi and Penicillium roqueforti isolates belonging to 3 genetically distinct populations.Finally, in 14 fungal species, laser nephelometry allowed a high-throughput monitoring of their growth after exposition to 3 different food preservatives and the determination of their associated minimal inhibitory concentrations.Overall, the obtained results demonstrate the usefulness of alternative techniques for identification and characterization of food spoilage fungi.; Les moisissures d’altération sont à l’origine de pertes alimentaires et économiques importantes et certaines espèces peuvent présenter un danger pour la santé humaine et animale avec la production de mycotoxines. Dans ce contexte, la maîtrise de la qualité et de la sécurité des aliments passe par une bonne connaissance des espèces impliquées. Cette connaissance repose sur une identification fiable et rapide et l’obtention d’informations sur les facteurs abiotiques impactant leur développement, tels que les conservateurs, largement utilisés dans l’industrie. Dans ce cadre, les objectifs de thèse étaient de développer l’utilisation de la spectrométrie de masse MALDI-TOF pour l’identification des moisissures et d’évaluer son application à la résolution de complexe d’espèces et au typage, et enfin d’évaluer la néphélométrie laser pour mesurer en haut-débit leur croissance en présence de conservateurs. Dans un premier temps, une base de données robuste a été construite avec près de 6500 spectres correspondant à 136 espèces fongiques. Dans un deuxième temps, la technique MALDI-TOF a été appliquée avec succès à la différenciation de 23 espèces du complexe Aspergillus section Flavi et a permis de différencier des isolats de Penicillium roqueforti appartenant à 3 populations génétiquement différenciées. Enfin, la néphélométrie laser a permis un suivi haut-débit de la croissance de 14 espèces fongiques d’altération en présence de 3 conservateurs et ainsi d’obtenir des informations sur les concentrations minimales inhibitrices de ces derniers. Ces travaux ont démontré l’applicabilité de techniques alternatives permettant d’identifier et de caractériser les moisissures d’altération.

Spoilage fungi represent a major cause of food and economic losses and certain species, which may produce mycotoxins, may also pose a threat to human and animal health. Thus, food safety and quality management relies notably on a good knowledge of the involved species. This knowledge is notably based on their fast and reliable identification and on the study of abiotic factors affecting their growth such as food preservatives, which are commonly used in the food industry. In this context, the objectives of this PhD. thesis were to develop MALDI-TOF mass spectrometry for mold identification and to evaluate its potential for species complex differentiation and strain typing, and finally, to evaluate the use of laser nephelometry to monitor fungal growth in the presence of food preservatives.First, a robust database was developed with 6500 spectra corresponding to 136 spoilage fungi. Then, MALDI-TOF MS was successfully applied to differentiate 23 species of Aspergillus section Flavi and Penicillium roqueforti isolates belonging to 3 genetically distinct populations.Finally, in 14 fungal species, laser nephelometry allowed a high-throughput monitoring of their growth after exposition to 3 different food preservatives and the determination of their associated minimal inhibitory concentrations.Overall, the obtained results demonstrate the usefulness of alternative techniques for identification and characterization of food spoilage fungi., Les moisissures d’altération sont à l’origine de pertes alimentaires et économiques importantes et certaines espèces peuvent présenter un danger pour la santé humaine et animale avec la production de mycotoxines. Dans ce contexte, la maîtrise de la qualité et de la sécurité des aliments passe par une bonne connaissance des espèces impliquées. Cette connaissance repose sur une identification fiable et rapide et l’obtention d’informations sur les facteurs abiotiques impactant leur développement, tels que les conservateurs, largement utilisés dans l’industrie. Dans ce cadre, les objectifs de thèse étaient de développer l’utilisation de la spectrométrie de masse MALDI-TOF pour l’identification des moisissures et d’évaluer son application à la résolution de complexe d’espèces et au typage, et enfin d’évaluer la néphélométrie laser pour mesurer en haut-débit leur croissance en présence de conservateurs. Dans un premier temps, une base de données robuste a été construite avec près de 6500 spectres correspondant à 136 espèces fongiques. Dans un deuxième temps, la technique MALDI-TOF a été appliquée avec succès à la différenciation de 23 espèces du complexe Aspergillus section Flavi et a permis de différencier des isolats de Penicillium roqueforti appartenant à 3 populations génétiquement différenciées. Enfin, la néphélométrie laser a permis un suivi haut-débit de la croissance de 14 espèces fongiques d’altération en présence de 3 conservateurs et ainsi d’obtenir des informations sur les concentrations minimales inhibitrices de ces derniers. Ces travaux ont démontré l’applicabilité de techniques alternatives permettant d’identifier et de caractériser les moisissures d’altération.

The immunoglobulin class of circulating immune complexes (IC) was examined in 35 patients with Behçet's syndrome (BS) according to the HLA phenotype of these patients. Cold precipitable complexes were separated and assayed by laser nephelometry and polyethylene glycol precipitable complexes were assayed by rocker immuno-electrophoresis for IgG, IgA and C3. The results suggest that in patients with BS, HLA-B12 is associated with an increased proportion of IgG and a decreased proportion of IgA and C3 in their immune complexes, whilst the converse was found with HLA-B5 or DRw2. The ratio of IgA:IgG in immune complexes was therefore low in patients with HLA-B12 but high in those with HLA-B5 or DRw2. We suggest that a linkage may exist between HLA and gene(s) determining the immunoglobulin class, in response to antigenic stimulation or the site of antigen localization.

The performance of the Alfred 60/AST device, an automated bacterial culture device which uses laser nephelometry to detect and quantify bacterial growth, was evaluated. The instrument is effective at screening negative samples and is more reliable at detecting bacteria than yeasts. Microscopy can be used to reduce the false-negative numbers.

Development of predictive in vitro surrogate methods for traditional approaches assessing bioavailability and pharmacokinetics of lead compounds must be made to both keep pace with highthroughput (HT) lead identification and to mitigate the high costs associated with progression of compounds with poor chances of developmental success. Indeed opportunities for improvement still exist in the lead optimization phase versus the lead identification phase, where HT methodologies have been nearly optimized. Review of examples, limitations, and development of high-throughput microtiterplate-based assays for evaluating metabolic liabilities, such as in vitro radiometric and fluorometric assays for inhibition of cytochrome P450 (CYP) activity, determination of stability of a compound in liver microsomes, or cloned CYPs coupled to reconstituting systems are described. Parallel approaches to improve speed, resolution, sample preparation, as well as data analysis using LC/MS and LC/MS/MS approaches and technologies to assess compound integrity and biotransformation by automation and multiplexing are also discussed. Realization of the benefits in automation of cell-based models for determining drug permeability to predict drug absorption are still hampered by bottlenecks in analytical analysis of compounds. The implementation and limitations of surrogate physiochemical methods for passive adsorption such as immobilized artificial membranes (IAM) and parallel artificial membrane permeation assays (PAMPA), and compound solubility by laser nephelometry are reviewed as well. Additionally, data from a high-throughput 96-well equilibrium dialysis device, showing good correlation to classical methods, is presented. Finally, the impact of improvements in these downstream bottlenecks in lead optimization and preclinical drug discovery are discussed in this review.

OBJECTIVE beta-Trace protein (beta-TP) is an immunological marker for the detection of cerebrospinal fluid traces. The aim of the study was to evaluate the predictive values of a new research assay for beta-TP. METHODS A total of 154 specimens from patients with otorrhea or rhinorrhea were investigated for cerebrospinal fluid (CSF) by use of a laser-nephelometric assay for beta-TP. Samples were obtained between January 1994 and November 2000. A sample was reported to be positive for CSF when the beta-TP concentration was more than 6 mg/L. Case evaluations were performed retrospectively and tabulated for indication, clinical course, additional investigations, surgical procedure, and follow-up. RESULTS beta-TP was detected in 16 specimens; 138 samples were negative for beta-TP with a value less than 3 mg/L. One sample was suggestive of CSF traces at 4.6 mg/L. In correlation with the clinical course, the intraoperative findings, intraoperative visualization with sodium fluorescein, high-resolution computed tomography of the paranasal sinuses or the petrous bone, computed tomographic cisternography, magnetic resonance imaging, and radionuclide cisternography, there was no false-positive result. On four occasions, false-negative results occurred, with an overall accuracy of 0.974. The beta-TP test had a negative predictive value of 0.971 and a positive predictive value of 1. CONCLUSION Analysis of betas-TP via the nephelometric assay is a valuable and reliable test in cranial base surgery for the identification of CSF.

The determination of aqueous solubility in a high-throughput screening environment is invaluable in the selection of the most promising potential drug candidates. We describe a fast method based on laser nephelometry that can determine the solubility of potential drug candidates (usually from combinatorial chemistry) supplied as dimethyl sulfoxide (DMSO) solutions in 96-well plates. In the sample, the percent DMSO is kept constant allowing direct comparison of results. The nephelometric method has been shown to produce results equivalent to those produced by an HPLC method and to be largely unaffected by colored solutions.

This article describes the development of a multifunctional prototype which supplies ultrasound to a sample in a Petri dish, between 18 KHz and 30KHz frequencies, with intensities from 0.3 mW/m2 to 1.5 mW/m2. This device guarantees sample survival with a micro-incubating system, operated by a control system and temperature monitoring. In addition, the prototype detects changes regarding the microbiological process in question, with a self-based laser-nephelometry system. The user would be able to choose parameters values mentioned, for a certain experiment, and analyze output data tables, images and statistics generated by a computer application specially designed to present this data., Este artículo describe el desarrollo de un prototipo multifuncional que suministra ultrasonido a una muestra en una placa de Petri, entre frecuencias de 18 KHz y 30 KHz, con intensidades de 0.3 mW / m2 a 1.5 mW / m2. Este dispositivo garantiza la supervivencia de la muestra con un sistema de microincubación, operado por un sistema de control y monitoreo de temperatura. Además, el prototipo detecta cambios con respecto al proceso microbiológico en cuestión, con un sistema de nefelometría láser con base propia. El usuario podría elegir los valores de los parámetros mencionados, para un determinado experimento, y analizar tablas de datos de salida, imágenes y estadísticas generadas por una aplicación informática especialmente diseñada para presentar estos datos.

International audience; By contrast with photometry (i.e., the measurement of light transmitted through a particle suspension), nephelometry is a direct method of measuring light scattered by particles in suspension. Since the scattered light intensity is directly proportional to the suspended particle concentration, nephelometry is a promising method for recording microbial growth and especially for studying filamentous fungi, which cannot be efficiently investigated through spectrophotometric assays. We describe herein for the first time a filamentous fungi-tailored procedure based on microscale liquid cultivation and automated nephelometric recording of growth, followed by extraction of relevant variables (lag time and growth rate) from the obtained growth curves. This microplate reader technique is applicable for the evaluation of antifungal activity and for large-scale phenotypic profiling.

The authors have tested two formulas by means of which the filtrated and the intraocularly (intrathecally) synthesized fraction of particular protein can be distinguished. The laser nephelometric method was applied to determine the serum (IgG, IgA, IgM, C3, albumin), aqueous humor (IgA, IgM, albumin) and CSF (IgG, IgM, C3, albumin) protein values in 42 subjects to test two models in physiological conditions. It was confirmed that the best results were achieved using the formula after Stambuk. Computer simulation performed to compare the formulas after Stambuk and Reiber & Felgenhauer showed a strong correlation (r greater than 0.98) between the calculated filtrated fractions of both models in a wide range of different barrier permeabilities. Laser nephelometry proved to be a useful method to determine low IgA and IgM values in aqueous humor.

We investigated serum concentration of C-reactive protein (CRP) and measures of adiposity in 30 patients with type 2 diabetes mellitus (15 male, 15 female) and 30 age and sex-matched apparently healthy subjects.CRP concentration was determined by laser nephelometry (BN II Analyzer) and CardioPhase high-sensitivity CRP (DADE BEHRING) was used as reagent which consists of polystyrene particles coated with mouse monoclonal antibodies to CRP.Results have shown that serum CRP concentration in patients with type 2 diabetes mellitus was statistically significantly higher compared to control group of healthy subjects (p

In this thesis chitosan and ß-cyclodextrin were used as candidates in medical textile applications. The antimicrobial activity of dissolved chitosan was evaluated quantitatively against both bacteria and fungi by different methods. The activity increased by increasing the concentration of chitosan. As a result, chitosan was applied as antimicrobial finishing agent to cotton by different anchor groups. Bacterial activity of chitosan-treated cotton was measured by tetrazolium/formazan test method. A water soluble chitosan derivative, namely carboxymethyl chitosan (CMCTS)was synthesized and characterized. CMCTS was applied to pre-cationized cotton forming ionically crosslinked cotton. The improvement in physical properties of cotton combined with a moderate antibacterial activity was achieved. In dentistry, chitosan films were applied to Molloplast® B, soft liner based material for dentures to treat the denture stomatitis. Also ß-cyclodextrin was used in pharmaceutical applications. Two antimycotic drugs were complexed by ß-cyclodextrin. The drug delivery and the solubility of the complexes were measured and evaluated by the laser nephelometry in microtiter plate in-vitro.

The physics principle of registration immune reaction by light scattering methods is concerned. The operation of laser nephelometry for measuring antigen-antibody reaction is described. The technique of obtaining diagnostic and immune reactions of interaction latex agglutination for diphtheria determination is described.

Laser nephelometry (LN) is a rapid and very sensitive method for simultaneous determination of albumin, immunoglobulins, C(3)c and C-4 in diluted serum and paired cerebrospinal fluid (CSF) samples. It is very useful in routine analyses. Determination of C(3)c and C-4 covers classical as well as alternative pathways of complement activation. In CSF, they are mostly derived from and related to serum values. Under physiological conditions, the addition of intrathecal C, synthesis is likely. The incidence of complement activation within CSF is also influenced by the method of choice (native molecules, activation products and complexes, inhibitors) and the mode of interpretation of results according to the functional state of the blood-brain barrier (BBB). Calculation of indexes and the modified Reiber's graph method are valid means of detection of complement activation within CSF. Complement activation within CSF was confirmed in 36% (111/302) of neurological patients examined ; in 55% (48/87) of patients with inflammatory and demyelinating diseases, in 40% (37/94) of patients with CNS infections and complications, in 33% (4/12) of patients with motor neuron diseases, in 27% (11/40) of patients with spinal cord compression and sequelae, in 25% (8/32) of patients with neoplastic disease, and in 17% (6/37) of patients with cerebrovascular accidents.

Summary: Concentrations of immunoglobuli ns were determined by laser nephelometry and, for comparison, by single radial immunodiffusion assay and a sandwich enzyme-linked immunosorbent assay (ELISA) in plasma samples of normal dogs. Correlation between methods was high, although the mean IgM concentrations determined by laser nephelometry were significantly higher than those determined by the single radial immunodirrusion assay or ELISA. In contrast, the mean IgA concentrations were significantly lower than those determined by ELISA. Laser nephelometry was the most reproducible method. The between-assay coefficient of variation was 4.12% for IgG, 6.98% for IgM and 6.35% for IgA. Laser nephelometry and ELISA showed similar accuracies, and both were more accurate than single radial immunodirrusion. Finally, laser nephelometry was much more sensitive than single radial immunodifrusion, but less sensitive than the ELISA method. In conclusion, results of the three methods were in general comparable. The higher range of linearity, precision and accuracy of laser nephelometry, and the availability of automated nephelometers make this the method of choice for quantifying canine plasma immunoglobulins.

The viscous properties of blood are of major importance for circulation in vessels with diameters less than 100 mm. The disturbance of microcirculation is observed in many pathologies. The blood viscosity under low shear rates is determined by the aggregation ability of erythrocytes. So, an important problem of hemorheology is the determination of the durability of aggregates, their sizes, and the aggregation kinetics. To measure these parameters the nephelometric technique is widely applied in different modifications enabling the registration of the backscattered or the transmitted light intensity. We present here the results of our study of peculiarities of the (dis)aggregation kinetics in relation to different pathologies: psoriatic arthritis, inherited hypercholesteremia, inherited homoglobinopathy, and some others. These results show the high diagnostic potentials of laser nephelometric technique.© (1993) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Methods of optical shifting spectroscopy and laser nephelometry may be used to register antigen-antibody reaction. Usage of laser nephelometry in serodiagnostics, characteristics of a specific step in humoral immune reaction in infections, postinfectious and postinoculation processes response and immunity tension in some infectious diseases were investigated.© (1993) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The effects of laser radiation on human organism are experimentally shown to be identifiable from optical (absorption K and extinction coefficients, mean cosine of the phase function of scattering, and value q/E, where qequals1/3(1- ) and temporal statistical characteristics of light being multiple scattered by blood.© (1993) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Laser backscattering nephelometry has been used to study dynamic properties of aggregating and nonaggregating suspensions of erythrocytes and of the whole blood in shear flows. The exponential dependences of total amplitude of aggregation on the hematocrit and on the shear rate have been found and the characteristic rapid and slow aggregation times have been estimated. A possibility to evaluate the shear diffusion coefficient for a suspension of nonaggregating erythrocytes has been demonstrated. 1.© (1991) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The effects of detergents on antigens and allergens of birch and timothy grass pollen extracts, codfish, hen egg-white, cat dander and house dust mite were investigated by crossed immunoelectrophoresis (CIE), crossed radio-immunoelectrophoresis (CRIE), radio-allergosorbent test (RAST) inhibition and quantitative precipitation test using laser nephelometry. Nine household cleaning solutions and five chemical detergents were tested. Higher concentrations than recommended for regular cleaning purposes were used as no influence on the antigenic and allergenic activities could be detected using the usual concentrations. Soft soap, guanidine hydrochloride and sodium lauryl sulphate induced most modifications of the antigens and allergens tested. None of the detergents totally destroyed the antigenic and allergenic activities of the selected material, even when used in concentrations up to 10 times that recommended. The materials used for carpet cleaning would not be able to impose any denaturing effects on allergens left on carpets after cleaning.

Orosomucoid was evaluated by laser-nephelometry in 1790 sera collected from 1170 newborns. Within-run precision (CV) was 2.1 to 4.2%, between-run 2.9 to 5.2%. Results correlated well with radial immunodiffusion (r = 0.989). Results can be obtained within 1 h. Orosomucoid concentrations in serum at birth range from 130 to 200 mg/L and are influenced by gestational age during the first two days of postnatal life. Thereafter, the values increase very rapidly in the first week of life, concentrations being the same as in adults by about 10 months. In 66 of 78 cases of severe bacterial infections, orosomucoid concentrations were above normal. Evidently, serum orosomucoid constitutes an useful index in diagnosis and monitoring of bacterial infections in the neonatal period.

Prothrombin antigen concentration was evaluated by means of laser nephelometry in 10 patients on coumarin therapy, in 17 patients with cirrhosis of the liver, and in four patients with congenital hypo- or dysprothrombinemias. The average values obtained were 46.4, 37.7, and 35.6%, respectively, for anticoagulated, cirrhotic, and congenitally abnormal patients. These values correlated well with those obtained by means of electroimmunoassay (Laurell) and immunodiffusion (Mancini) methods. Similarly, satisfactory results were obtained in eight normal subjects. Multiple evaluations at different incubation times, also allowed the authors to construct kinetic curves of the interaction between antigen and antibody. However, an abnormal kinetic curve was demonstrated only for coumarin-treated patients.

The quantitative determination of glycoproteins and lectins by laser nephelometry is based on the fact that these two compounds form a complex comparable to the antigen-antibody precipitate. Due to the high sensitivity and reliability of this technique less than 25 pmol of glycoprotein or 4.5 pmol of lectin can be determined, thus providing a suitable method (a) to investigate the specificity of different lectins for certain carbohydrate residues, (b) to study the mode of interaction, and (c) to explore mammalian tissue for still-unknown lectin-like substances.

A nephelometric procedure for quantitative measurement of glycohemoglobin (Hb A1) was developed, evaluated, and compared with the semi-quantitative mini-column chromatographic procedure. Hb A1 was purified from human red cell hemolystate by Bio-Rex 70 ion-exchange liquid chromatography and was used for standards and immunization. The antisera raised in rabbits showed high cross-reactivities with normal human hemoglobin (Hb A). The latter was separated by mixing 25 μl of patients' hemolystate with 2 ml ion-exchange resin suspension for 15 minutes. One hundred microiters of the supernatant was incubated with 900 μl antiserum (dilution 1:50) for 45 minutes at room temperature. Samples were then read on a laser nephelometer. The sensitivity of the assay was found to be 0.1 mg Hb A1. The intraassay relative standard deviation (RSD) was 5.6% and the interassay RSD was 6.5%.

An adaptation of the nephelometric assay for serum immunoglobulins has been developed for detection and quantitation of extracellular immunoglobulins in cultures of lymphoblastoid cell lines. This assay employs the standard equipment for laser nephelometry and commercial reagents for immunoglobulin quantitation. By adjusting dilutions of controls and sample volumes of culture supernatants, amounts of IgG and IgM below 1 μg/ml can be detected in culture supernatants. At concentrations between 1 and 4 μg/ml, day-to-day and within-run variations for IgM assays were 16 and 11% respectively. The possibility of measuring immunoglobulins secreted by cell lines by conventional laser nephelometry opens several areas of application in the study of the functional activity of B cells and of cell-cell interactions.

A simple technique using high performance liquid gel permeation chromatography for determination of IgG in cerebrolspinal fluid (CSF) is described. Accurate determination of IgG was achieved by precolumn derivation with fluorescein isothiocyanate-conjugated anti-human IgG Fab, a substance that readily yields a fluorescent immune complex. Immune complexes separated as a well-delineated peak in the column void volume, and were measured by the fluorescence of the column eluates (Ex = 490 nm, Em = 520 nm. The results were reproducible with a deviation of less than 2% and a good linear relationship over the range of 2.0–24.5 mg/dl. The IgG concentration assayed by the column method correlated well with values obtained by laser nephelometry.

Serum immunoglobulin concentrations were determined on sera from 298 healthy children aged six months to six years using the Hyland laser nephelometer PDQ system. Age-specific 95% reference ranges for serum IgG, IgA and IgM are presented; considerable care has been taken to ensure statistical validity of the reference ranges. The wide range of values in children under two years suggest that measuring immunoglobulin concentrations in this age group is of little value in diagnosing immunodeficiency.