Measurement of 5-day biochemical oxygen demand without sample dilution or bacterial and nutrient enhancement

A direct method for measuring the 5-day biochemical oxygen demand (BODS) of aquaculture samples that does not require sample dilution or bacterial and nutrient enrichment was evaluated. The regression coefficient (R-2) between the direct method and the standard method for the analyses of 32 samples from catfish ponds was 0.996. The slope of the regression line did not differ from 1.0 or the Y-intercept from 0.0 at P = 0.05. Thus, there was almost perfect agreement between the two methods. The control limits (three standard deviations of the mean) for a standard solution containing 15 mg/L each of glutamic acid and glucose were 17.4 and 20.4 mg/L. The precision of the two methods, based on eight replicate analyses of four pond water samples did not differ at P = 0.05. (c) 2005 Elsevier B.V All rights reserved.

Evaluation of Inductively Coupled Plasma Mass Spectrometry for Determining Ca, Cu, Fe, Mg, Mn, Se and Zn in Bovine Semen Samples using a Simple Sample Dilution Method

A simple and fast method for the determination of Ca, Cu, Fe, Mg, Mn, Se and Zn in bovine semen by quadrupole inductively coupled plasma spectrometry (q-ICP-MS) is described. Prior to analysis, samples (200 mu L) were diluted 1:50 in a solution containing 0.01% v/v Triton (R) X-100 and 0.5% v/v nitric acid and directly analyzed by ICP-MS. The limits of detection of the method are 0.3, 0.03, 0.2, 0.04, 0.04, 0.03 and 0.03 mu g L-1 for Ca-44, Cu-63, Fe-57, Mg-24, Zn-64, Se-82 and Mn-55, respectively. For purposes of comparison and method validation, four ordinary bovine semen samples were directly analyzed by ICP-MS and by flame atomic absorption spectrometry (FAAS) or graphite furnace atomic absorption spectrometry (GF AAS), with no statistical difference between the techniques at the 95% level when applying the t-test. Then, the proposed method was applied in the determinations of Ca, Cu, Fe, Mg, Mn, Se and Zn in collected samples of bovine semen from different breeds, which are used in reproduction programs and artificial insemination.

Evaluation of inductively coupled plasma mass spectrometry for determining Ca, Cu, Fe, Mg, Mn, Se and Zn in bovine semen samples using a simple sample dilution method

A simple and fast method for the determination of Ca, Cu, Fe, Mg, Mn, Se and Zn in bovine semen by quadrupole inductively coupled plasma spectrometry (q-ICP-MS) is described. Prior to analysis, samples (200 µL) were diluted 1:50 in a solution containing 0.01% v/v Triton® X-100 and 0.5% v/v nitric acid and directly analyzed by ICP-MS. The limits of detection of the method are 0.3, 0.03, 0.2, 0.04, 0.04, 0.03 and 0.03 µg L-1 for 44Ca, 63Cu, 57Fe, 24Mg, 64Zn, 82Se and 55Mn, respectively. For purposes of comparison and method validation, four ordinary bovine semen samples were directly analyzed by ICP-MS and by flame atomic absorption spectrometry (FAAS) or graphite furnace atomic absorption spectrometry (GF AAS), with no statistical difference between the techniques at the 95% level when applying the t-test. Then, the proposed method was applied in the determinations of Ca, Cu, Fe, Mg, Mn, Se and Zn in collected samples of bovine semen from different breeds, which are used in reproduction programs and artificial insemination.

Simple Serological Assays for Detecting Rice Tungro Viruses

An attempt was made to produce sensitive and specific polyclonal antisera against the viruses causing rice tungro disease, and to assess their potential for use in simple diagnostic tests. Using a multiple, sequential injection procedure, seven batches of polyclonal antisera against rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV) were produced. These were characterized for their sensitivity and specificity using ring-interface precipitin test and double antibody sandwich (DAS) ELISA. Thirty-one weeks after the first immunization, antiserum batch B6b for RTBV showed the highest ring interface titer (DEP = 1:1920). For RTSV, batches S3, S4b and S5b all had similar titres (DEP = 1:640). In DAS-ELISA, however, significant differences among purified antisera (IgG) batches were observed only at IgG dilution of 10-3. At that dilution, IgGB4b showed the greatest sensitivity, while IgGS3 showed greatest sensitivity for RTSV. When all IgG batches were tested against 11 tungro field isolates (dual RTBV-RTSV infections) at sample dilution of 1:10, IgGB4b and IgGB6b for RTBV and IgGS3 and IgGS6b for RTSV performed equally well. However, after cross adsorption with healthy plant extracts in a specially prepared healthy plant-Sepharose affinity column, only IgGB6b could be used specifically to detect RTBV in a simple tissue-print assay.

Investigation of the airborne submicrometer particles emitted by dredging vessels using a plume capture method

This paper presents a method for investigating ship emissions, the plume capture and analysis system (PCAS), and its application in measuring airborne pollutant emission factors (EFs) and particle size distributions. The current investigation was conducted in situ, aboard two dredgers (Amity: a cutter suction dredger and Brisbane: a hopper suction dredger) but the PCAS is also capable of performing such measurements remotely at a distant point within the plume. EFs were measured relative to the fuel consumption using the fuel combustion derived plume CO2. All plume measurements were corrected by subtracting background concentrations sampled regularly from upwind of the stacks. Each measurement typically took 6 minutes to complete and during one day, 40 to 50 measurements were possible. The relationship between the EFs and plume sample dilution was examined to determine the plume dilution range over which the technique could deliver consistent results when measuring EFs for particle number (PN), NOx, SO2, and PM2.5 within a targeted dilution factor range of 50-1000 suitable for remote sampling. The EFs for NOx, SO2, and PM2.5 were found to be independent of dilution, for dilution factors within that range. The EF measurement for PN was corrected for coagulation losses by applying a time dependant particle loss correction to the particle number concentration data. For the Amity, the EF ranges were PN: 2.2 - 9.6 × 1015 (kg-fuel)-1; NOx: 35-72 g(NO2).(kg-fuel)-1, SO2 0.6 - 1.1 g(SO2).(kg-fuel)-1and PM2.5: 0.7 – 6.1 g(PM2.5).(kg-fuel)-1. For the Brisbane they were PN: 1.0 – 1.5 x 1016 (kg-fuel)-1, NOx: 3.4 – 8.0 g(NO2).(kg-fuel)-1, SO2: 1.3 – 1.7 g(SO2).(kg-fuel)-1 and PM2.5: 1.2 – 5.6 g(PM2.5).(kg-fuel)-1. The results are discussed in terms of the operating conditions of the vessels’ engines. Particle number emission factors as a function of size as well as the count median diameter (CMD), and geometric standard deviation of the size distributions are provided. The size distributions were found to be consistently uni-modal in the range below 500 nm, and this mode was within the accumulation mode range for both vessels. The representative CMDs for the various activities performed by the dredgers ranged from 94-131 nm in the case of the Amity, and 58-80 nm for the Brisbane. A strong inverse relationship between CMD and EF(PN) was observed.

Development of a rapid multiplexed assay for the direct screening of antimicrobial residues in raw milk

Antimicrobial residues found to be present in milk can have both health and economic impacts. For these reasons, the widespread routine testing of milk is required. Due to delays with sample handling and test scheduling, laboratory-based tests are not always suited for making decisions about raw material intake and product release, especially when samples require shipping to a central testing facility. Therefore, rapid on-site screening tests that can produce results within a matter of minutes are required to facilitate rapid intake and product release processes. Such tests must be simple for use by non-technical staff. There is increasing momentum towards the development and implementation of multiplexing tests that can detect a range of important antimicrobial residues simultaneously. A simple in situ multiplexed planar waveguide device that can simultaneously detect chloramphenicol, streptomycin and desfuroylceftiofur in raw dairy milk, without sample preparation, has been developed. Samples are simply mixed with antibody prior to an aliquot being passed through the detection cartridge for 5 min before reading on a field-deployable portable instrument. Multiplexed calibration curves were produced in both buffer and raw milk. Buffer curves, for chloramphenicol, streptomycin and desfuroylceftiofur, showed linear ranges (inhibitory concentration (IC)₂₀–IC₈₀) of 0.1–0.9, 3–129 and 12–26 ng/ml, whilst linear range in milk was 0.13–0.74, 11–376 and 2–12 ng/ml, respectively, thus meeting European legislated concentration requirements for both chloramphenicol and streptomycin, in milk, without the need for any sample preparation. Desfuroylceftiofur-contaminated samples require only simple sample dilution to bring positive samples within the range of quantification. Assay repeatability and reproducibility were lower than 12 coefficient of variation (%CV), whilst blank raw milk samples (n = 9) showed repeatability ranging between 4.2 and 8.1 %CV when measured on all three calibration curves.

Validation of a novel ELISA for measurement of electronegative low-density lipoprotein

Background: Oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathogenesis of atherosclerosis. LDL(-) is present in blood plasma of healthy subjects and at higher concentrations in diseases with high cardiovascular risk, such as familial hypercholesterolemia or diabetes. Methods: We developed and validated a sandwich ELISA for LDL(-) in human plasma using two monoclonal antibodies against LDL(-) that do not bind to native LDL, extensively copper-oxidized LDL or malondialdehyde-modified LDL. The characteristics of assay performance, such as limits of detection and quantification, accuracy, inter- and intra-assay precision were evaluated. The linearity, interferences and stability tests were also performed. Results: The calibration range of the assay is 0.625-20.0 mU/L at 1: 2000 sample dilution. ELISA validation showed intra- and inter- assay precision and recovery within the required limits for immunoassays. The limits of detection and quantification were 0.423 mU/L and 0.517 mU/L LDL(-), respectively. The intra- and inter- assay coefficient of variation ranged from 9.5% to 11.5% and from 11.3% to 18.9%, respectively. Recovery of LDL(-) ranged from 92.8% to 105.1%. Conclusions: This ELISA represents a very practical tool for measuring LDL(-) in human blood for widespread research and clinical sample use. Clin Chem Lab Med 2008; 46: 1769-75.

Amphotericin B Microemulsion Reduces Toxicity and Maintains the Efficacy as an Antifungal Product

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Indirect cathodic-stripping voltammetric determination of ceftazidime as a mercury salt

At accumulation potentials close to +0.1 V at a hanging mercury drop electrode, ceftazidime is accumulated at pH 9.5, probably in a hydrolysed or otherwise chemically altered form, in an anodic process to give an adsorbed mercury salt. The accumulation of this mercury salt allows the indirect cathodic-stripping voltammetric determination of ceftazidime using the reduction peak of the mercury salt at -0.70 V. The high sensitivity of the method coupled with high sample dilution allows ceftazidime to be determined in milk samples at the 28 mu g ml(-1) level without prior separation. In order to determine lower levels of ceftazidime in milk (ca. 10 ng ml(-1)) a separation process would be required. (C) 1998 Elsevier B.V. B.V. All rights reserved.

Uso da padronização interna em espectrometria de absorção atômica em forno de grafite na determinação de 'SE' e 'NI' em bebidas não alcoólicas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Comparison of flow cytometry and indirect immunofluorescence assay in the diagnosis and cure criterion after therapy of American tegumentary leishmaniasis by anti-live Leishmania (Viannia) braziliensis immunoglobulin G

The aim of this study was to compare the techniques of indirect immunofluorescence assay (IFA) and flow cytometry to clinical and laboratorial evaluation of patients before and after clinical cure and to evaluate the applicability of flow cytometry in post-therapeutic monitoring of patients with American tegumentary leishmaniasis (ATL). Sera from 14 patients before treatment (BT), 13 patients 1 year after treatment (AT), 10 patients 2 and 5 years AT were evaluated. The results from flow cytometry were expressed as levels of IgG reactivity, based on the percentage of positive fluorescent parasites (PPFP). The 1:256 sample dilution allowed us to differentiate individuals BT and AT. Comparative analysis of IFA and flow cytometry by ROC (receiver operating characteristic curve) showed, respectively, AUC (area under curve) = 0.8 (95% CI = 0.64–0.89) and AUC = 0.90 (95% CI = 0.75–0.95), demonstrating that the flow cytometry had equivalent accuracy. Our data demonstrated that 20% was the best cut-off point identified by the ROC curve for the flow cytometry assay. This test showed a sensitivity of 86% and specificity of 77% while the IFA had a sensitivity of 78% and specificity of 85%. The after-treatment screening, through comparative analysis of the technique performance indexes, 1, 2 and 5 years AT, showed an equal performance of the flow cytometry compared with the IFA. However, flow cytometry shows to be a better diagnostic alternative when applied to the study of ATL in the cure criterion. The information obtained in this work opens perspectives to monitor cure after treatment of ATL.

The collapse of linear polyelectrolyte chains in a poor solvent: When does a collapsing polyelectrolyte collect its counter ions?

The collapse of linear polyelectrolyte chains in a poor solvent: When does a collapsing polyelectrolyte collect its counter ions? The collapse of polyions in a poor solvent is a complex system and is an active research subject in the theoretical polyelectrolyte community. The complexity is due to the subtle interplay between hydrophobic effects, electrostatic interactions, entropy elasticity, intrinsic excluded volume as well as specific counter-ion and co-ion properties. Long range Coulomb forces can obscure single molecule properties. The here presented approach is to use just a small amount of screening salt in combination with a very high sample dilution in order to screen intermolecular interaction whereas keeping intramolecular interaction as much as possible (polyelectrolyte concentration cp ≤ 12 mg/L, salt concentration; Cs = 10^-5 mol/L). This is so far not described in literature. During collapse, the polyion is subject to a drastic change in size along with strong reduction of free counterions in solution. Therefore light scattering was utilized to obtain the size of the polyion whereas a conductivity setup was developed to monitor the proceeding of counterion collection by the polyion. Partially quaternized PVP’s below and above the Manning limit were investigated and compared to the collapse of their uncharged precursor. The collapses were induced by an isorefractive solvent/non-solvent mixture consisting of 1-propanol and 2-pentanone, with nearly constant dielectric constant. The solvent quality for the uncharged polyion could be quantified which, for the first time, allowed the experimental investigation of the effect of electrostatic interaction prior and during polyion collapse. Given that the Manning parameter M for QPVP4.3 is as low as lB / c = 0.6 (lB the Bjerrum length and c the mean contour distance between two charges), no counterion binding should occur. However the Walden product reduces with first addition of non solvent and accelerates when the structural collapse sets in. Since the dielectric constant of the solvent remains virtually constant during the chain collapse, the counterion binding is entirely caused by the reduction in the polyion chain dimension. The collapse is shifted to lower wns with higher degrees of quaternization as the samples QPVP20 and QPVP35 show (M = 2.8 respectively 4.9). The combination of light scattering and conductivity measurement revealed for the first time that polyion chains already collect their counter ions well above the theta-dimension when the dimensions start to shrink. Due to only small amounts of screening salt, strong electrostatic interactions bias dynamic as well as static light scattering measurements. An extended Zimm formula was derived to account for this interaction and to obtain the real chain dimensions. The effective degree of dissociation g could be obtained semi quantitatively using this extrapolated static in combination with conductivity measurements. One can conclude the expansion factor a and the effective degree of ionization of the polyion to be mutually dependent. In the good solvent regime g of QPVP4.3, QPVP20 and QPVP35 exhibited a decreasing value in the order 1 > g4.3 > g20 > g35. The low values of g for QPVP20 and QPVP35 are assumed to be responsible for the prior collapse of the higher quaternized samples. Collapse theory predicts dipole-dipole attraction to increase accordingly and even predicts a collapse in the good solvent regime. This could be exactly observed for the QPVP35 sample. The experimental results were compared to a newly developed theory of uniform spherical collapse induced by concomitant counterion binding developed by M. Muthukumar and A. Kundagrami. The theory agrees qualitatively with the location of the phase boundary as well as the trend of an increasing expansion with an increase of the degree of quaternization. However experimental determined g for the samples QPVP4.3, QPVP20 and QPVP35 decreases linearly with the degree of quaternization whereas this theory predicts an almost constant value.

Evaluation of the multi-element capabilities of collision/reaction cell inductively coupled plasma - mass spectrometry in wine analysis

This work explores the multi-element capabilities of inductively coupled plasma - mass spectrometry with collision/reaction cell technology (CCT-ICP-MS) for the simultaneous determination of both spectrally interfered and non-interfered nuclides in wine samples using a single set of experimental conditions. The influence of the cell gas type (i.e. He, He+H2 and He+NH3), cell gas flow rate and sample pre-treatment (i.e. water dilution or acid digestion) on the background-equivalent concentration (BEC) of several nuclides covering the mass range from 7 to 238 u has been studied. Results obtained in this work show that, operating the collision/reaction cell with a compromise cell gas flow rate (i.e. 4 mL min−1) improves BEC values for interfered nuclides without a significant effect on the BECs for non-interfered nuclides, with the exception of the light elements Li and Be. Among the different cell gas mixtures tested, the use of He or He+H2 is preferred over He+NH3 because NH3 generates new spectral interferences. No significant influence of the sample pre-treatment methodology (i.e. dilution or digestion) on the multi-element capabilities of CCT-ICP-MS in the context of simultaneous analysis of interfered and non-interfered nuclides was observed. Nonetheless, sample dilution should be kept at minimum to ensure that light nuclides (e.g. Li and Be) could be quantified in wine. Finally, a direct 5-fold aqueous dilution is recommended for the simultaneous trace and ultra-trace determination of spectrally interfered and non-interfered elements in wine by means of CCT-ICP-MS. The use of the CCT is mandatory for interference-free ultra-trace determination of Ti and Cr. Only Be could not be determined when using the CCT due to a deteriorated limit of detection when compared to conventional ICP-MS.

Characterisation of a harvesting step for monoclonal antibodies from mammalian cell culture

The focus of this research was defined by a poorly characterised filtration train employed to clarify culture broth containing monoclonal antibodies secreted by GS-NSO cells: the filtration train blinded unpredictably and the ability of the positively charged filters to adsorb DNA from process material was unknown. To direct the development of an assay to quantify the ability of depth filters to adsorb DNA, the molecular weight of DNA from a large-scale, fed-batch, mammalian cell culture vessel was evaluated as process material passed through the initial stages of the purification scheme. High molecular weight DNA was substantially cleared from the broth after passage through a disc stack centrifuge and the remaining low molecular weight DNA was largely unaffected by passage through a series of depth filters and a sterilising grade membrane. Removal of high molecular weight DNA was shown to be coupled with clarification of the process stream. The DNA from cell culture supernatant showed a pattern of internucleosomal cleavage of chromatin when fractionated by electrophoresis but the presence of both necrotic and apoptotic cells throughout the fermentation meant that the origin of the fragmented DNA could not be unequivocally determined. An intercalating fluorochrome, PicoGreen, was elected for development of a suitable DNA assay because of its ability to respond to low molecular weight DNA. It was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing pertinent monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 89.0 % of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples. Application of the fluorescence based assay resulted in characterisation of the physical parameters governing adsorption of DNA by various positively charged depth filters and membranes in test solutions and the DNA adsorption profile of the manufacturing scale filtration train. Buffers that reduced or neutralised the depth filter or membrane charge, and those that impeded hydrophobic interactions were shown to affect their operational capacity, demonstrating that DNA was adsorbed by a combination of electrostatic and hydrophobic interactions. Production-scale centrifugation of harvest broth containing therapeutic protein resulted in the reduction of total DNA in the process stream from 79.8 μg m1-1 to 9.3 μg m1-1 whereas the concentration of DNA in the supernatant of pre-and post-filtration samples had only marginally reduced DNA content: from 6.3 to 6.0 μg m1-1 respectively. Hence the filtration train was shown to ineffective in DNA removal. Historically, blinding of the depth filters had been unpredictable with data such as numbers of viable cells, non-viable cells, product titre, or process shape (batch, fed-batch, or draw and fill) failing to inform on the durability of depth filters in the harvest step. To investigate this, key fouling contaminants were identified by challenging depth filters with the same mass of one of the following: viable healthy cells, cells that had died by the process of apoptosis, and cells that had died through the process of necrosis. The pressure increase across a Cuno Zeta Plus 10SP depth filter was 2.8 and 16.5 times more sensitive to debris from apoptotic and necrotic cells respectively, when compared to viable cells. The condition of DNA released into the culture broth was assessed. Necrotic cells released predominantly high molecular weight DNA in contrast to apoptotic cells which released chiefly low molecular weight DNA. The blinding of the filters was found to be largely unaffected by variations in the particle size distribution of material in, and viscosity of, solutions with which they were challenged. The exceptional response of the depth filters to necrotic cells may suggest the cause of previously noted unpredictable filter blinding whereby a number of necrotic cells have a more significant impact on the life of a depth filter than a similar number of viable or apoptotic cells. In a final set of experiments the pressure drop caused by non-viable necrotic culture broths which had been treated with DNase I or benzonase was found to be smaller when compared to untreated broths: the abilities of the enzyme treated cultures to foul the depth filter were reduced by 70.4% and 75.4% respectively indicating the importance of DNA in the blinding of the depth filter studied.

High resolution mass spectrometry for quantitative analysis and untargeted screening of algal toxins in mussels and passive samplers

Measurement of marine algal toxins has traditionally focussed on shellfish monitoring while, over the last decade, passive sampling has been introduced as a complementary tool for exploratory studies. Since 2011, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been adopted as the EU reference method (No.15/2011) for detection and quantitation of lipophilic toxins. Traditional LC-MS approaches have been based on low-resolution mass spectrometry (LRMS), however, advances in instrument platforms have led to a heightened interest in the use of high-resolution mass spectrometry (HRMS) for toxin detection. This work describes the use of HRMS in combination with passive sampling as a progressive approach to marine algal toxin surveys. Experiments focused on comparison of LRMS and HRMS for determination of a broad range of toxins in shellfish and passive samplers. Matrix effects are an important issue to address in LC-MS; therefore, this phenomenon was evaluated for mussels (Mytilus galloprovincialis) and passive samplers using LRMS (triple quadrupole) and HRMS (quadrupole time-of-flight and Orbitrap) instruments. Matrix-matched calibration solutions containing okadaic acid and dinophysistoxins, pectenotoxin, azaspiracids, yessotoxins, domoic acid, pinnatoxins, gymnodimine A and 13-desmethyl spirolide C were prepared. Similar matrix effects were observed on all instruments types. Most notably, there was ion enhancement for pectenotoxins, okadaic acid/dinophysistoxins on one hand, and ion suppression for yessotoxins on the other. Interestingly, the ion selected for quantitation of PTX2 also influenced the magnitude of matrix effects, with the sodium adduct typically exhibiting less susceptibility to matrix effects than the ammonium adduct. As expected, mussel as a biological matrix, quantitatively produced significantly more matrix effects than passive sampler extracts, irrespective of toxin. Sample dilution was demonstrated as an effective measure to reduce matrix effects for all compounds, and was found to be particularly useful for the non-targeted approach. Limits of detection and method accuracy were comparable between the systems tested, demonstrating the applicability of HRMS as an effective tool for screening and quantitative analysis. HRMS offers the advantage of untargeted analysis, meaning that datasets can be retrospectively analysed. HRMS (full scan) chromatograms of passive samplers yielded significantly less complex data sets than mussels, and were thus more easily screened for unknowns. Consequently, we recommend the use of HRMS in combination with passive sampling for studies investigating emerging or hitherto uncharacterised toxins.