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At least 487 records · Page 27Linked to original sources
PubMed2002

Quantification of microcystin-producing cyanobacteria and E. coli in water by 5'-nuclease PCR.

AIMS: 5'-Nuclease (real-time, quantitative) PCR methodologies were developed and applied as diagnostic tools for the detection of microcystin-producing cyanobacteria and Escherichia coli in water. METHODS AND RESULTS: PCR was used to detect regions of the lacZ gene in E. coli, and the microcystin synthetase gene in microcystin-producing cyanobacteria. In environmental water samples, natural inhibitors to PCR were effectively removed with a prefiltration step and an EDTA wash. A lower detection limit of 10 cells ml(-1) was obtained with endpoint PCR detection. 5'-Nuclease PCR was used for microbial quantification of 1 ml inoculated water samples. We were able to detect down to three copies of our target genes per sample within about 2 h (post-DNA isolation) for both E. coli and microcystin-producing cyanobacteria. CONCLUSIONS: 5'-Nuclease PCR offers a rapid and sensitive method of bacterial quantification in water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: 5'-Nuclease PCR can be adopted as an effective diagnostic tool for monitoring microbiological water quality, through coliform quantification, and detection of other waterborne microbial pathogens.

Cyanobacteria↗
PubMed2001

Quantification of pyrethroid insecticides from treated bednets using a mosquito recombinant glutathione S-transferase.

Recombinant glutathione S-transferase (agGST1-6) from the malaria vector mosquito Anopheles gambiae Giles (Diptera: Culicidae) was expressed in Escherichia coli using a pET3a vector system. The expressed enzyme was biochemically active with reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Activity of agGST1-6 with GSH and CDNB was inhibited to different degrees by both alpha-cyano and non-alpha-cyano pyrethroid insecticides. This inhibition was used to develop an assay for quantification of pyrethroids. Standard curves of insecticide concentration against percentage of enzyme inhibition or volume of iodine solution were established by spectrophotometry and iodine volumetric titration, respectively, for permethrin and deltamethrin. These assays allowed estimation of pyrethroid concentrations both spectrophotometrically and visually. For the residue assay of each insecticide, a cut-off point of 50% of the initial pyrethroid impregnation concentration was used, which should differentiate between biologically active and inactive treated bednets. The cross-reactivity of the primary permethrin photodegradants (3-phenoxyalcohol and 3-phenoxybenzoic acid) with the recombinant agGST1-6 was assayed in the same system. No agGST1-6 inhibition by the insecticide metabolites was observed, suggesting that the system is unaffected by primary permethrin metabolites and will accurately measure insecticide parent compound concentrations. The estimated pyrethroid insecticide concentrations, given spectrophotometrically and by iodine titration assay, were comparable to those obtained by direct HPLC quantification of residues extracted from bednets. Hence, it should be relatively easy to adapt this method to produce a test kit for residue quantification in the field.

Animals↗
PubMed2000

Migration of mononuclear cells in the modified Boyden chamber as evaluated by DNA quantification and flow cytometry.

In vitro migration of mononuclear cells in the modified Boyden chamber was evaluated using flow cytometry and DNA quantification (Hoechst 33258) of all adherent and nonadherent cells. The effects of different membrane pore sizes, cell concentrations and incubation times were studied. Pore sizes of 3 and 5 microm resulted in a reduction in the number of nonadherent cells compared with a pore size of 8 microm. Reducing the incubation time from 60 to 40 and 20 min resulted in too few migrating monocytes for analysis by flow cytometry. Flow cytometry showed that both monocytes and lymphocytes migrated and adhered to the membrane when using peripheral blood mononuclear cells (PBMC) to study monocyte migration. Migration of lymphocytes under these conditions is a novel observation. A substantial number of migrated cells could be identified by flow cytometry and quantified by DNA measurement as nonadherent below the membrane. Samples of synovial fluid (n = 49) and plasma (n = 133) as chemoattractants analysed in triplicate resulted in mean coefficient of variation (CV) values of 11 and 9%, respectively. Variation from assay to assay on the same day, using N-formyl-methionyl-leucyl-phenylanine (fMLP) 10(-7) M as chemoattractant resulted in a CV of 13%. Day-to-day variation, using fMLP 10(-7) M as chemoattractant and the same well on three different days, resulted in a CV of 21%. These results were obtained using a pore size of 5 microm, a PBMC concentration of 3 x 10(6)/ml and 60 min of incubation. The combination of DNA quantification and flow cytometry thus allowed characterization and quantification of subsets of migrating adherent as well as nonadherent mononuclear cells.

Antigens, CD↗
PubMed2001

Identification and quantification of anti-D, -C and -G in alloimmunized pregnant women.

Our objectives were to investigate possible overestimation of maternal anti-D due to co-existing anti-C and/or anti-G, and to confirm the presence of anti-D in plasma presumed to contain anti-D+C. We investigated 96 samples (from 22 antenatal patients and 74 blood donors) initially identified as containing anti-D+C using routine investigation procedures. Anti-D quantification was performed using an Astoria Pacific International 300 (API 300) continuous flow analyser with R1R1 and R2R2 reagent red cells. Where possible, samples were tested manually using a rare D+, C-, G- cell, to confirm the presence of anti-D. Fifty-two of 96 samples (11/22 antenatal patients and 41/74 blood donors) gave >50% higher anti-D quantification results with R1R1 cells than with R2R2 cells. Anti-D was not detected using manual techniques in 16 of 73 samples tested (10/22 antenatal patients and 6/51 blood donors). Anti-D quantification using R1R1 reagent red cells may cause inaccurate estimation of anti-D levels, when anti-C and/or anti-G are present. Indeed, a significant number of cases, where apparent anti-D+C is identified, may contain only anti-C+G and lack an anti-D component. This may in turn lead to a failure to administer prophylactic anti-D immunoglobulin to RhD negative patients in cases where anti-D is not present, putting these patients at risk from immunization with possible consequences to future pregnancies.

Blood Grouping and Crossmatching↗
PubMed1999

GC/MS analysis of anandamide and quantification of N-arachidonoylphosphatidylethanolamides in various brain regions, spinal cord, testis, and spleen of the rat.

Anandamide [N-arachidonoylethanolamide (NAE)] was initially isolated from porcine brain and proposed as an endogenous ligand for cannabinoid receptors in 1992. Accumulating evidence has now suggested that, in the tissue, NAE is generated from N-arachidonoylphosphatidylethanolamides (N-ArPEs) by phosphodiesterase. In this study a sensitive and specific procedure was developed to quantify NAE and N-ArPE, including organic solvent extraction, reverse-phase C-18 cartridge separation, derivatization, and gas chromatography/mass spectrometry (GC/MS) analysis. NAE is converted by a two-step derivatization procedure to a pentafluorobenzoyl ester followed by pentafluoropropionyl acylation. Quantification was performed by isotope dilution GC/MS using deuterium-labeled NAE (NAE-2H8) as an internal standard. The same chemical derivatization was applicable to N-ArPE quantification. The separated N-ArPE fractions were converted by a two-step cleavage/derivatization procedure into the pentafluorobenzoyl ester of NAE and then to its pentafluoropropionyl amide. The derivative was quantified by GC/MS using deuterium-labeled 1,2-[2H8]dioleoyl-sn-glycero-3-phospho(arachidonoyl)ethanolamid e as an internal standard. Using these methods, we have found that endogenous NAE levels in rat brain, spleen, testis, liver, lung, and heart were below the level of quantification achievable (0.1 pmol/mg of protein) but that N-ArPE is readily quantifiable and is widely distributed in the rat CNS with the highest level in the spinal cord. The striatum, hippocampus, and accumbens contain intermediate concentrations of N-ArPE, whereas the value is lowest in the cerebellum.

Animals↗
PubMedDate not supplied

Future directions in dialysis quantification.

The influence of dialysis prescription on outcome is well established, and currently the amount of dialysis prescribed is based on small molecular weight toxin removal as represented by the clearance of urea. The "normalized dose of dialysis" (Kt/V(urea)) concept is well established. Most techniques for dialysis quantification require that blood samples be taken at the beginning and after the completion of dialysis. The postdialysis sample, however, gives cause for concern because of the "rebound phenomenon" due to nonuniform distribution of urea among body compartments. Blood samples give "indirect" measures of dialysis quantification. Thus direct urea concentration measurements in dialysate may be superior in urea kinetic modeling and these may be made "real time" during dialysis. It is with real-time monitoring that future advances in dialysis quantification will take place. These will be of two types. The first will analyze blood water or dialysate samples for urea content multiple times throughout the treatment; the second will assess the on-line clearance of urea using surrogate molecules such as sodium chloride, the clearance being determined by conductivity measurements. On-line urea monitoring is based on the action of urease on urea in a water solution and measurement of the resultant ammonium ions, which are measured directly by a specific electrode or indirectly by conductivity changes. Differences in blood-side versus dialysate-side urea monitors exist which reflect the parameters they can provide, but with both, the standard urea kinetic measurements of Kt/V and nPCR (nPNA) are easily obtainable. A range of additional parameters can be derived from dialysate-side monitoring such as "whole-body Kt/V," "pretreatment urea mass" and "whole-body urea clearance," which are worthy of future studies to determine their roles in adequacy assessment. Conductivity clearance measurements are made by examining the conductivity differences between dialysate inlet and outlet measured at two different dialysate inlet concentrations. This allows for the calculation of the electrolyte (ionic) dialysance, which is equal to the "effective" urea clearance, that is, the clearance that takes into account recirculation effects that reduce hemodialysis efficiency. The continuous reading of effective ionic clearance will allow an average value for K to be obtained for that dialysis, and hence the parameter K x t as an indication of dialysis dose is easily and accurately obtained for every treatment. The conductivity technology is cheap and rugged, and thus expanded use can be expected. Urea monitors have an inherent cost and require maintenance, and perhaps will remain researchers' tools for the present. The methodologies can complement each other; the addition of an accurate and independent value for K to dialysate based urea monitoring is like having simultaneous blood- and dialysate-side monitoring, and allows further increase in measurable parameters.

Humans↗
PubMed2000

Rapid quantification of left ventricular function and mass using transoesophageal three-dimensional echocardiography: validation of a method that uses long-axis cutplanes.

AIMS: Despite its proven superiority compared to conventional echocardiographic techniques, three-dimensional (3D) echocardiography has not gained widespread acceptance in clinical medicine for the quantification of left ventricular volumes, function and mass. This is mainly due to the large, time-consuming process of data analysis. We sought to validate a new method that enables the accurate quantification of the left ventricle in a clinically acceptable short period of time. METHODS AND RESULTS: Left ventricular volumes, ejection fraction and mass were determined in 44 patients using 3D echocardiography. The 3D echocardiographic data sets were analysed: (i) using the conventional 'summation of slices' algorithm (slice thickness 5 and 10mm), which is based on the analysis of the 3D reconstructed left ventricle in short-axis cross-sections; and (ii) using the new method which is based on the analysis of the 3D reconstructed left ventricle in long-axis images. In each patient measurements were repeated using 3, 6, 7, 8, 9, 12 and 15 long-axis images. For all volumetric measurements there was a continuous reduction of measurement variability using increasing numbers of long-axis images. The use of more than nine long-axis images for volumes, and eight long-axis images for ejection fraction and mass, did not result in a further reduction of variability. The analysis time for volumes and masses averaged less than 5 min for the long-axis method using nine component images, compared to 20-43 min for the short-axis method. CONCLUSION: 3D echocardiography combined with a novel method based on the analysis of long-axis cross-section allows accurate quantification of left ventricular volumes, function and mass in a clinically acceptable short period of time. In the future, the combination of a real-time 3D echocardiographic acquisition technique with this analysis method should have important implications for the introduction of 3D echocardiography in clinical practice.

Echocardiography, Three-Dimensional↗
PubMed2002

Clinical utility of total HCV core antigen quantification: a new indirect marker of HCV replication.

Hepatitis C virus (HCV) RNA detection, viral load quantification, and HCV genotyping are widely used in clinical practice. Recently, the availability of an anticore antigen (Ag) monoclonal antibody allowed development of an enzyme-linked immunosorbent assay (ELISA) detecting and quantifying total HCV core Ag in peripheral blood of HCV-infected patients. The aims of the present study were to investigate the biologic significance of this new marker in HCV infection, to establish the intrinsic performance of the current assay, and to determine its potential utility in the management of HCV-infected patients. A panel of infected sera calibrated to the World Health Organization International Standard and 657 serum samples from infected patients receiving antiviral treatment were studied. We showed that total HCV core Ag quantification is an accurate, precise, and specific indirect marker of HCV replication. We estimated that 1 pg/mL of total HCV core Ag is equivalent to approximately 8,000 HCV RNA international units (IU)/mL, although minor between-patient differences may exist. In conclusion, total HCV core Ag quantification can be used in the various indications of viral load monitoring, including the evaluation of baseline viral load before therapy, the assessment of the virologic response to antiviral treatment, and the study of early viral kinetics during therapy. Nevertheless, the total HCV core Ag assay cannot be used as a marker of viral replication for HCV RNA values below 20,000 IU/mL, limiting its use in the monitoring of late events during and after antiviral treatment.

Biomarkers↗
PubMed2003

[Prospective analysis of quantification of contrast media enhanced power Doppler sonography of equivocal breast lesions].

PURPOSE: To evaluate the potential diagnostic value of quantification of contrast enhanced power Doppler sonography in breast lesions. MATERIALS AND METHODS: Before and after bolus application of 300 mg/ml of the contrast agent Levovist (Schering, Germany), 76 randomly selected equivocal breast lesions (37 benign, 39 malignant) underwent power Doppler sonography. The data were acquired with a 7.5 MHz linear array transducer (Sonoline Versa Pro and Sonoline Elegra Plus, Siemens, Germany). Following postprocessing digitalization, color pixel density (CPD) and dynamic contrast enhancement were calculated and compared using different regions of the tumor (entire lesion, center and periphery). RESULTS: A measurable pixel signal prior to the application of the contrast agent was observed in 12 of 37 benign and in 18 of 39 malignant lesions (32.4 % and 46.2 %, respectively). After administration of the contrast agent, the lesions with measurable signal increased to 20 of 37 (54 %) and 25 of 39 (64.1 %), respectively. No statistically significant difference of dignity-related signal intensity could be verified before or after administration of the contrast agent. Maximum CPD and relative increase in pixel density after contrast medium was appreciably higher in the center of malignant lesions than in corresponding regions of benign lesions. CONCLUSION: Quantification of color pixel density is rather nonspecific for the discrimination of equivocal breast lesions and not suitable for clinical routine. Signal detection and quantification of color coded pixels enable comparison of dynamic contrast enhancement.

Adult↗
PubMed1997

[CT quantification of pleuropulmonary lesions in severe thoracic trauma].

PURPOSE: Computed quantification of the extent of pleuropulmonary trauma by CT and comparison with conventional chest x-ray--Impact on therapy and correlation with mechanical ventilation support and clinical outcome. METHOD: In a prospective trial, 50 patients with clinically suspicious blunt chest trauma were evaluated using CT and conventional chest x-ray. The computed quantification of ventilated lung provided by CT volumetry was correlated with the consecutive artificial respiration parameters and the clinical outcome. RESULTS: We found a high correlation between CT volumetry and artificial ventilation concerning maximal pressures and inspiratory oxygen concentration (FiO2, Goris-Score) (r = 0.89, Pearson). The graduation of thoracic trauma correlated highly with the duration of mechanical ventilation (r = 0.98, Pearson). Especially with regard to atelectases and lung contusions CT is superior compared to conventional chest x-ray; only 32% and 43%, respectively, were identified by conventional chest x-ray. CONCLUSION: CT allows rapid classification and quantification of pulmonary lesions after thoracic trauma and provides higher sensitivity and reliability. Because of the great correlation with the extent of artificial respiration in respect of duration and pressure, prognosis of the individual patient, as well as a differential therapy, appear possible.

Adolescent↗
PubMed2003

Normal values of left ventricular systolic and diastolic function derived from signal-averaged acoustic quantification waveforms: a multicenter study.

Automated border-detection techniques such as acoustic quantification have proven accurate and useful for quantifying left ventricular (LV) function. We acquired LV acoustic quantification waveforms from the parasternal short-axis window in 140 healthy patients in the age range of 16 to 78 years. Signal-averaged waveforms were analyzed for parameters of systolic and diastolic performance. The average fractional area change was 54 +/- 12%, and there were no significant changes in LV systolic function in the age range studied. There were significant changes in diastolic parameters with aging. The percentage of contribution to total LV filling occurring during atrial filling nearly tripled during the 6 decades studied, from 13% in the youngest cohort to 36% in the eighth decade of life. This study provides normal reference values for systolic and diastolic parameters of LV function determined from signal-averaged acoustic quantification waveforms acquired from the parasternal short-axis view in adult and adolescent patients over a wide age range.

Adolescent↗
PubMed2001

Quantification of aortic regurgitation by real-time 3-dimensional echocardiography in a chronic animal model: computation of aortic regurgitant volume as the difference between left and right ventricular stroke volumes.

BACKGROUND: The accuracy of conventional 2-dimensional echocardiographic and Doppler techniques for the quantification of valvular regurgitation remains controversial. In this study, we examined the ability of real-time 3-dimensional (RT3D) echocardiography to quantify aortic regurgitation by computing aortic regurgitant volume as the difference between 3D echocardiographic-determined left and right ventricular stroke volumes in a chronic animal model. METHODS: Three to 6 months before the study, 6 sheep underwent surgical incision of one aortic valve cusp to create aortic regurgitation. During the subsequent open chest study session, a total of 25 different steady-state hemodynamic conditions were examined. Electromagnetic (EM) flow probes were placed around the main pulmonary artery and ascending aorta and balanced against each other to provide reference right and left ventricular stroke volume (RVSV and LVSV) data. RT3D imaging was performed by epicardial placement of a matrix array transducer on the volumetric ultrasound system, originally developed at the Duke University Center for Emerging Cardiovascular Technology. During each hemodynamic steady state, the left and right ventricles were scanned in rapid succession and digitized image loops stored for subsequent measurement of end-diastolic and end-systolic volumes. Left and right ventricular stroke volumes and aortic regurgitant volumes were then calculated and compared with reference EM-derived values. RESULTS: There was good correlation between RT3D left and right ventricular stroke volumes and reference data (r = 0.83, y = 0.94x + 2.6, SEE = 9.86 mL and r = 0.63, y = 0.8x - 1.0, SEE = 5.37 mL, respectively). The resulting correlation between 3D- and EM-derived aortic regurgitant volumes was at an intermediate level between that for LVSV and that for RVSV (r = 0.80, y = 0.88x + 7.9, SEE = 10.48 mL). RT3D tended to underestimate RVSV (mean difference -4.7 +/- 5.4 mL per beat, compared with -0.03 +/- 9.7 mL per beat for the left ventricle). There was therefore a small overestimation of aortic regurgitant volume (4.7 +/- 10.4 mL per beat). CONCLUSION: Quantification of aortic regurgitation through the computation of ventricular stroke volumes by RT3D is feasible and shows good correlation with reference flow data. This method should also be applicable to the quantification of other valvular lesions or single site intracardiac shunts where a difference between right and left ventricular cavity stroke volumes is produced.

Animals↗
PubMed2003

Real-time three-dimensional color doppler evaluation of the flow convergence zone for quantification of mitral regurgitation: Validation experimental animal study and initial clinical experience.

BACKGROUND: Pitfalls of the flow convergence (FC) method, including 2-dimensional imaging of the 3-dimensional (3D) geometry of the FC surface, can lead to erroneous quantification of mitral regurgitation (MR). This limitation may be mitigated by the use of real-time 3D color Doppler echocardiography (CE). Our objective was to validate a real-time 3D navigation method for MR quantification. METHODS: In 12 sheep with surgically induced chronic MR, 37 different hemodynamic conditions were studied with real-time 3DCE. Using real-time 3D navigation, the radius of the largest hemispherical FC zone was located and measured. MR volume was quantified according to the FC method after observing the shape of FC in 3D space. Aortic and mitral electromagnetic flow probes and meters were balanced against each other to determine reference MR volume. As an initial clinical application study, 22 patients with chronic MR were also studied with this real-time 3DCE-FC method. Left ventricular (LV) outflow tract automated cardiac flow measurement (Toshiba Corp, Tokyo, Japan) and real-time 3D LV stroke volume were used to quantify the reference MR volume (MR volume = 3DLV stroke volume - automated cardiac flow measurement). RESULTS: In the sheep model, a good correlation and agreement was seen between MR volume by real-time 3DCE and electromagnetic (y = 0.77x + 1.48, r = 0.87, P <.001, delta = -0.91 +/- 2.65 mL). In patients, real-time 3DCE-derived MR volume also showed a good correlation and agreement with the reference method (y = 0.89x - 0.38, r = 0.93, P <.001, delta = -4.8 +/- 7.6 mL). CONCLUSIONS: real-time 3DCE can capture the entire FC image, permitting geometrical recognition of the FC zone geometry and reliable MR quantification.

Adult↗
PubMed2006

Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum.

The postsynaptic density (PSD) of central excitatory synapses is essential for postsynaptic signaling, and its components are heterogeneous among different neuronal subtypes and brain structures. Here we report large scale relative and absolute quantification of proteins in PSDs purified from adult rat forebrain and cerebellum. PSD protein profiles were determined using the cleavable ICAT strategy and LC-MS/MS. A total of 296 proteins were identified and quantified with 43 proteins exhibiting statistically significant abundance change between forebrain and cerebellum, indicating marked molecular heterogeneity of PSDs between different brain regions. Moreover we utilized absolute quantification strategy, in which synthetic isotope-labeled peptides were used as internal standards, to measure the molar abundance of 32 key PSD proteins in forebrain and cerebellum. These data confirm the abundance of calcium/calmodulin-dependent protein kinase II and PSD-95 and reveal unexpected stoichiometric ratios between glutamate receptors, scaffold proteins, and signaling molecules in the PSD. Our data also demonstrate that the absolute quantification method is well suited for targeted quantitative proteomic analysis. Overall this study delineates a crucial molecular difference between forebrain and cerebellar PSDs and provides a quantitative framework for measuring the molecular stoichiometry of the PSD.

Animals↗
PubMed2005

Difference in mass analysis using labeled lysines (DIMAL-K): a new, efficient proteomic quantification method applied to the analysis of astrocytic secretomes.

Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 microg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins.

Animals↗
PubMed2005

Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition.

Relative quantification methods have dominated the quantitative proteomics field. There is a need, however, to conduct absolute quantification studies to accurately model and understand the complex molecular biology that results in proteome variability among biological samples. A new method of absolute quantification of proteins is described. This method is based on the discovery of an unexpected relationship between MS signal response and protein concentration: the average MS signal response for the three most intense tryptic peptides per mole of protein is constant within a coefficient of variation of less than +/-10%. Given an internal standard, this relationship is used to calculate a universal signal response factor. The universal signal response factor (counts/mol) was shown to be the same for all proteins tested in this study. A controlled set of six exogenous proteins of varying concentrations was studied in the absence and presence of human serum. The absolute quantity of the standard proteins was determined with a relative error of less than +/-15%. The average MS signal responses of the three most intense peptides from each protein were plotted against their calculated protein concentrations, and this plot resulted in a linear relationship with an R(2) value of 0.9939. The analyses were applied to determine the absolute concentration of 11 common serum proteins, and these concentrations were then compared with known values available in the literature. Additionally within an unfractionated Escherichia coli lysate, a subset of identified proteins known to exist as functional complexes was studied. The calculated absolute quantities were used to accurately determine their stoichiometry.

Blood Proteins↗
PubMed2000

Antigen quantification as in vitro alternative for potency testing of inactivated viral poultry vaccines.

Routine batch control of licensed inactivated viral vaccines for poultry usually includes a potency assay as a measure of vaccine efficacy. Potency assays often consist of vaccination-challenge experiments in the target species or in laboratory animals. Instead of measuring the protection of vaccinated animals against virulent pathogens, the serological response after vaccination can be quantified for some vaccines. In vitro antigen quantification assays would be attractive alternatives for the current potency assays because the time and costs involved could be greatly reduced and animal use could be avoided. Such in vitro assays will only be acceptable when the correlation between results and efficacy or potency has been demonstrated convincingly. The results of our studies on antigen quantification assays indicate that, in principle, quantification of viral antigens from inactivated oil-adjuvanted vaccines is feasible and reproducible using specially developed antigen capture ELISAs in combination with specific software for statistical analysis of the ELISA data. We have developed methods to quantify the haemagglutination-neuraminidase (HN) and fusion (F) proteins of Newcastle disease virus (NDV), the viral protein 3 (VP3) of the infectious bursal disease virus (IBDV), and the spike-1 (S1) protein of the infectious bronchitis virus (IBV). Vaccination experiments with inactivated ND vaccines indicate that the in vitro quantified HN- or F-proteins of NDV are reliable indicators of the serological response after vaccination.

Animals↗
PubMed2004

Migration from can coatings: part 3. Synthesis, identification and quantification of migrating epoxy-based substances below 1000 Da.

Bisphenol A-derived glycidyl ethers as well as its reaction products with other lacquer components can migrate into the packed food from epoxy-based can coatings. A sensitive and selective method is presented using high-performance liquid chromatography coupled with ultraviolet light, fluorescence and electrospray ionization-mass selective detection for the identification and quantification of all migrants with a bisphenol A backbone and a molecular weight below 1000 Da, an estimated boundary for the absorption in the gastrointestinal tract. The identification of migrants was confirmed by microreactions of technical bisphenol A diglycidyl ether with solvents and phenols, which provided the fragmentation pattern of the mass selective detection and relative retentions of 42 different bisphenol A-related substances. It was shown by calibration of different isolated and synthesized bisphenol A derivatives that the fluorescence response relies on the amount of bisphenol A moiety in the respective molecule. Therefore, all migrating bisphenol A-related substances below 1000 Da were determined as bisphenol A diglycidyl ether equivalents using a calibration (fluorescence detection) of the commercially available bisphenol A diglycidyl ether monomer. The limit of quantification was set at 5 microg bisphenol A diglycidyl ether equivalents kg(-1) (or 0.8 microg dm(-2)). This method was validated for epoxy coatings (0.1 microg dm(-2) limit of detection and 24 microg bisphenol A-related substances below 1000 Da dm(-2) standard deviation, corresponding to 4.4% relative standard deviation). The quantification could be extended by combining the fluorescence response and structural information gained from the mass spectra, which provides more accurate results for each migrant. The calculation is based on the calibration of the bisphenol A chromophore content of the molecule. According to this method, the amount of migrating bisphenol A-related substances below 1000 Da in the acetonitrile extract (assuming a worst case) varied from about 0.4 to 0.7 mg dm(-2) in the examined coatings. The determined amounts comply with about 50% of the total migrate below 1000 Da.

Benzhydryl Compounds↗