Effects Of Assay Conditions Research Articles

Effect of assay conditions on the migration of phthalates from polyvinyl chloride cling films used for food packaging in México

A survey of cling-films commercially available in México was performed to identify/quantify the phthalate content and determine overall (OM) and specific migration using two types of cells. Of the 15 films acquired, 14 consisted of polyvinyl chloride. The only phthalate detected in five of the films was di(2-ethylhexyl) phthalate (DEHP), with a content of <0.1 % in three of them and that of 24.8 % and 17.7 % in the remaining two. Two other films contained di(2-ethylhexyl) terephthalate (DEHT) at 16.4 % and 1.5 %. OM of the seven films in aqueous and acidic food simulants was within the limit set by the European Union and MERCOSUR legislation and exceeded when tested in two-sided migration cells using a fatty food simulant. OM was lower when single-sided cells were used. Two films presented significant DEHP migration, while the others, containing non-detectable levels of DEHP or DEHT but high OM, were deduced to contain non-phthalate additives.

Colorimetric determination of cysteine by a paper-based assay system using aspartic acid modified gold nanoparticles.

Amethod is describedfor cysteine (Cys) determination on paper-based analytical devices using aspartic acid modified gold nanoparticles (Asp-AuNPs). The Asp-AuNPs were characterized by their size, zeta potential, and UV-visible absorption spectrum. After the addition of Cys, it will interact with Asp-AuNPs selectively and leads to the aggregation of Asp-AuNPs. A color change from red to blue can be observed on the paper-based analytical devices. The results were recorded using a cell phone and subsequently analyzed using the Photoshop software. The ratiometric color intensity at red channel and blue channel (Red/Blue) increased linearly in the range 99.9-998.7μM for Cys (R = 0.9984), and the limit of detection was 1.0μM. The effects of assay conditions have been investigated and are discussed. The Cys concentration was determined as (0.27 ± 0.02mM) in human plasma, and the recovery was from 99.2 to 101.1%. Graphical abstract Schematic representation of the paper-based assay system using aspartic acid modified gold nanoparticles (Asp-AuNPs). The ratiometric color intensity method was used for the cysteine (Cys) determination.

Investigation of the inhibition of eight major human cytochrome P450 isozymes by a probe substrate cocktail in vitro with emphasis on CYP2E1

1. A protocol has been developed and validated for the high-throughput screening of eight major human cytochrome P450 (CYP) isozymes inhibition (CYP 1A2, 2C9, 2C19, 2D6, 3A4, 2B6, 2C8 and 2E1) using an in vitro probe cocktail containing eight substrates by overcoming the unfavorable effect of assay conditions on CYP2E1 inhibition data.2. The cocktail consisting of selective probe substrates like tacrine (CYP1A2), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A4), bupropion (CYP2B6), paclitaxel (CYP2C8) and chlorzoxazone (CYP2E1) was incubated with human liver microsomes.3. The method was investigated by incubating well-known CYP inhibitors {alphanaphthoflavone (CYP1A2), sulfaphenazole (CYP2C9), N-3-benzylnirvanol (CYP2C19), quinidine (CYP2D6), ketoconazole (CYP3A4), ticlopidine (CYP2B6), quercetin (CYP2C8) and 4-methylpyrazole (CYP2E1)} with the substrate cocktail. A fast gradient liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for this study.4. The IC50 values determined for typical CYP inhibitors were reproducible and consistent with those in the literature. DMSO has significant effect and itself inhibits CYP2E1. DMSO should not exceed 0.1% for the determination of reliable CYP2E1 inhibition profile. This cocktail assay offers an efficient and robust method to determine the CYP450 isoforms inhibition profiles of large numbers of compounds in a quick turnaround time.

Measuring phenol oxidase and peroxidase activities with pyrogallol, l-DOPA, and ABTS: Effect of assay conditions and soil type

Microbial phenol oxidases and peroxidases mediate biogeochemical processes in soils, including microbial acquisition of carbon and nitrogen, lignin degradation, carbon mineralization and sequestration, and dissolved organic carbon export. Measuring oxidative enzyme activities in soils is more problematic than assaying hydrolytic enzyme activities because of the non-specific, free radical nature of the reactions and complex interactions between enzymes, assay substrates, and the soil matrix. We compared three substrates commonly used to assay phenol oxidase and peroxidase in soil: pyrogallol (PYGL, 1,2,3-trihydroxybenzene), l-DOPA (l-3,4-dihydroxyphenylalanine), and ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). We measured substrate oxidation in three soils across a pH gradient from 3.0 to 10.0 to determine the pH optimum for each substrate. In addition, we compared activities across 17 soils using the three substrates. In general, activities on the substrates followed the trend PYGL > l-DOPA > ABTS and were inversely related to substrate redox potential. PYGL and ABTS were not suitable substrates at pH > 5, and ABTS oxidation often declined with addition of peroxide to the assay. Absolute and relative oxidation rates varied widely among substrates in relation to soil type and assay pH. We also tested whether autoclaved or combusted soils could be used as negative controls for the influence of abiotic factors (e.g., soil mineralogy) on oxidative activity. However, neither autoclaving nor combustion produced reliable negative controls because substrate oxidation still occurred; in some cases, these treatments enhanced substrate oxidation rates. For broad scale studies, we recommend that investigators use all three substrates to assess soil oxidation potentials. For focused studies, we recommend evaluating substrates before choosing a single option, and we recommend assays at both the soil pH and a reference pH (e.g., pH 5.0) to determine the effect of assay pH on oxidase activity. These recommendations should contribute to greater comparability of oxidase potential activities across studies.

Isotope disequilibrium and mass spectrometric studies of inorganic carbon acquisition by phytoplankton

Given the need to assess potential effects of rising atmospheric CO2 on aquatic primary productivity, many studies have investigated the physiological mechanisms of inorganic carbon acquisition by a variety of phytoplankton species. Membrane inlet mass spectrometry (MIMS) has become the preferred methodological approach for laboratory experiments, whereas the 14C disequilibrium method has proven to be particularly useful for field studies. In the present investigation, we explicitly compare results of carbon acquisition measurements obtained with both of these approaches. Testing a range of phytoplankton species from different taxa, we show that both methods provide nearly identical results on the contribution of HCO3− and CO2 relative to net carbon fixation. In contrast, although both approaches yielded highly reproducible estimates for extracellular carbonic anhydrase activity, the results differed significantly between the two methods. By directly comparing these two leading methods, we provide experimental confirmation of key assumptions used for data interpretation and discuss possible effects of assay conditions. Our analysis highlights the individual strengths and weaknesses of different approaches.

Effects of assay conditions in life history experiments with Drosophila melanogaster

Abstract Selection experiments with Drosophila have revealed constraints on the simultaneous evolution of life history traits. However, the responses to selection reported by different research groups have not been consistent. Two possible reasons for these inconsistencies are (i) that different groups used different environments for their experiments and (ii) that the selection environments were not identical to the assay environments in which the life history traits were measured. We tested for the effect of the assay environment in life history experiments by measuring a set of Drosophila selection lines in laboratories working on life history evolution with Drosophila in Basel, Groningen, Irvine and London. The lines measured came from selection experiments from each of these laboratories. In each assay environment, we measured fecundity, longevity, development time and body size. The results show that fecundity measurements were particularly sensitive to the assay environment. Differences between assay and selection environment in the same laboratory or differences between assay environments between laboratories could have contributed to the differences in the published results. The other traits measured were less sensitive to the assay environment. However, for all traits there were cases where the measurements in one laboratory suggested that selection had an effect on the trait, whereas in other laboratories no such conclusion would have been drawn. Moreover, we provide good evidence for local adaptation in early fecundity for lines from two laboratories.

Evaluation of the decarbamylation process of cholinesterase during assay of enzyme activity

The activity of carbamylated cholinesterase increases continuously during assay, suggesting that progressive decarbamylation takes place. The following effects of assay conditions on the observed decarbamylation were studied: the effect of the sulfhydryl group of nitrobenzoate produced in the course of Ellman assay, the effect of substrate and the effect of sample dilution during assay. This study indicates that sample dilution is the main trigger to the decarbamylation observed during assay of cholinesterase activity. The process was described as a first-order reaction during which the inhibited enzyme gives place to the active form. Kinetic constants for decarbamylation of human pseudocholinesterase (EC 3.1.1.8) at 30°C were approximately 0.005 min −1 for dimethylcarbamates and 0.010 min −1 for monomethylcarbamates, when 1 mmol/l propionylthiocholine was used as substrate.

Effect of assay conditions on measurement of elastolytic activity of alveolar macrophages in culture and characterization with proteinase inhibitors

We have investigated whether varied assay conditions account for the conflicting reports on measured alastolytic activity of alveolar macrophages (AM) cultured in direct contact with the 3H-elastin substrate coated onto 16-mm wells in serum-containing media. The data indicate that measured elastolytic activity in this assay system was dependent on the amount of 3H-elastin/culture well. 3H-elastin >350 μg/well, in contrast to the ⩽200 μg/well commonly used in this assay system, resulted in optimal measurement of elastolytic activity that was linear with respect to culture time (up to 72 h examined) and was directly proportional to number of AM/well (up to 1.0 × 10 6 examined). The sensitivity of measured elastolytic activity to tissue inhibitor of metalloproteinases (TIMP) and to Z-phe-phe (a specific cysteine proteinase inhibitor) was not affected by amount of 3H-elastin/well, but appears to be dependent on the time period of AM culture. TIMP (at 5 μM, maximal dose examined) inhibited the measured elastolytic activity by 25% in 24-h cultures compared to 69% in 72-h cultures; Z-phe-phe (at 10 μM, dose at which maximal effect was obtained) inhibited the elastolytic activity by 45% in the 24-h cultures compared to 34% in the 72-h cultures. These findings indicate that differences in substrate levels and in culture time have a significant effect on the results obtained in measurement of AM-mediated elastolytic activity in culture, which may account for the conflicting reports in the literature. Thus standard optimal assay condition are required for valid interpretation of results of AM-mediated elastolytic activity measurements.

Characterization of Egg Yolk Antibodies for Detection and Quantification of Selenomonas ruminantium by Using an Enzyme-Linked Immunosorbent Assay

The specificity of polyclonal antibodies prepared against strains of Selenomonas ruminantium, the effect of assay conditions, and quantification of individual strains in mixed-cell suspensions of selenomonad strains were examined in this study. Whole-cell suspensions were prepared with pure cultures of S. ruminantium PC18, HD(4), GA192, and D. Each cell suspension was injected into a Leghorn laying hen, and polyclonal antibodies were harvested from eggs laid in week 3 or 7 following initial immunization. Antibodies made to the S. ruminantium strains readily discerned the homologous strain from the heterologous strains. Cross-reactivity among antibodies and the heterologous S. ruminantium strains ranged from 5 to 26%. Among non-S. ruminantium species, cross-reactivity of S. ruminantium antibodies was greatest with Selenomonas sputigena (3 to 34%) and Succinivibrio dextrinosolvens (0 to 37%). Antibodies made to strains GA192 and D were used to quantify a mixture of the two strains. Both antibodies responded to graded concentrations of the homologous antigen in the biculture mixtures in accord with the change in the direct cell counts for each strain (strain D, R = 0.92; strain GA192, R = 0.90). This enzyme-linked immunosorbent assay enabled concurrent and accurate quantification of two strains of S. ruminantium subsp. ruminantium in a mixed-cell suspension with a precision of much less than 1 order of magnitude.

The effect of assay conditions on the measurement of anti-elastase function in lung secretions

We have determined the effect of altering assay conditions on the observed neutrophil elastase inhibitory capacity in lung secretions from emphysematous patients with normal serum α 1 PI. α 1 PI, ALP and α 2-macroglobulin were detected in all samples. Measurement at low enzyme concentration (< 33.6 nmol/l) caused a 43% reduction in observed neutrophil elastase inhibitory capacity of sputum sol-phase, while inhibition by secretions in buffer without added NaC1 was 20% greater than in the presence of 0.2 mol/l NaCl. Increasing the concentration of the synthetic substrate Suc-[Ala] 3-pNA in the assay from 0.45 mmol/l to 7.2 mmol/l reduced the observed inhibitory capacity by 53% and the use of elastin-fluorescein gave lower results for inhibitory capacity than the Suc-[Ala] 3-pNA (median 0.26 mol neutrophil elastase/mol measured inhibitors (range 0–0.72) with elastin; 1.40 mol neutrophil elastase/mol measured inhibitors (0.80–3.21) with Suc-[Ala] 3-pNA). Assay conditions therefore greatly influence the results. In addition these findings suggest the presence of an additional inhibitor of neutrophil elastase in these secretions.

Effect of assay conditions on cross reactivity of digoxin-like immunoreactive substance(s) with radioimmunoassay kits.

One or more digoxin-like immunoreactive substances (DLIS), most frequently present in serum of premature and full-term neonates. cross react to various extents with different digoxin immunoassay kit reagents. Mostly, this variation is attributed to the relative cross reactivity of DLIS with the antiserum in each kit. However, modification of standard assay procedures for digoxin can also greatly alter the relative cross reactivity of DLIS. Using sequential RIA kit methods for digoxin by 20 to 60% relative to the standard equilibrium RIA mode. Cross reactivity was decreased still more if the concentrations of antiserum (binding-site concentration) and tracer (125 l-labeled digoxin) were decreased, and conversely. Serum samples containing only digoxin, analyzed by the modified method, consistently yielded results well comparable with those obtained with the manufacturers' recommended procedures. We describe use of the different responses to digoxin and DLIS of standard and sequential radioimmunoassays and use of simultaneous equation to calculate the concentration of DLIS (in digoxin equivalents) in digoxin-containing samples.

Effect of assay conditions and field exposure on urease activity associated with cereal residues

Abstract Urea fertilisers are often applied to soil with cereal residues on the surface. Although the urease properties of soils have been investigated, the urease activities associated with cereal residues have not been thoroughly studied. Two experiments were performed to develop an acceptable urease assay for cereal residues, and to determine the effect of field exposure and concomitant saprophytic colonization on urease levels. Two acceptable assays were found: 1) incubation in the presence of THAM‐H2SO4 buffer (pH 8), toluene, and urea, and 2) incubation only in the presence of urea. In both assays substrate concentration was 0.05 M, incubation temperature was 37°C, incubation length was 2 hours, and urea hydrolysis was determined by ammonium production. Although both assays were highly correlated (r2 = 0.992), the buffer method gave higher values. Subsequent investigations illustrated that urease activity associated with cereal residues is dramatically increased by saprophytic colonization after har...

Steroid receptor binding to nuclei: Effect of assay conditions on the integrity of chromatin

Various incubation conditions previously used in cell-free assays of steroid receptor binding to nuclei were evaluated on the basis of their effect on the integrity of the nuclear chromatin. Incubation of nuclei for 60 min in whole cytosol containing no added salt at 25°C resulted in significant DNA degradation and some proteolysis. This deterioration in the integrity of the chromatin increased its capacity to bind the [ 3H]-progesterone-receptor complex, possibly via exposure of previously “masked” acceptor sites. Decreasing the temperature and/or increasing the salt concentration reduced or eliminated these changes in the chromatin. Incubation of nuclei in an ammonium sulfate-fraction of the cytosol, which contained the partially purified progesterone receptor, resulted in no detectable deterioration of the chromatin integrity irrespective of the temperature (⩽25°C) or the added salt concentration (0–0.15 M KCl). These results suggest that some of the conflicting reports on the nature of the nuclear acceptor sites for steroid receptors could be due to the use of inappropriate assay conditions, which permit degradation of the nucleo-protein and result in altered levels of steroid receptor binding. (Steroid/ Progesterone/Nuclei/Chromatin.)

The Effect of Assay Conditions on the Recovery of Schistosomula from the Lungs of Normal and Resistant Rats Infected with Schistosoma mansoni

The recovery of schistosomula from lungs of normal and resistant Sprague-Dawley and Fischer rats was analyzed by varying the assay conditions. The effects of incubation medium, incubation time, and perfusion procedure on yields of schistosomula were significant and additive. Optimal assay conditions were established to recover schistosomula by the lung recovery assay. Differences were observed in the number of schistosomula recovered from lungs of normal and resistant rats; the assay was performed as a function of time after challenge infection. Differences in schistosomula recovered were significant on days 3 to 5 (Fischer) and days 3 to 6 (Sprague-Dawley); afterwards, no differences were observed in recoveries of schistosomula from lungs. Interpretations of these findings are disscused.

Microsomal cytochrome P-450 from the housefly, Musca domestica: Assay and spectral characterization

The effects of assay conditions on spectral characterization of microsomal cytochrome P-450 from insecticide-resistant (Fc) and insecticide-susceptible (CSMA) strains of the housefly, ( Musca domestica) were investigated. The specific activity of cytochrome P-450 was higher in microsomes prepared from isolated abdomens than in those from whole flies. Buffers of low molarity (<0·1 M) caused denaturation of cytochrome P-450 and the optimum pH values, in terms of cytochrome P-450 content, were pH 7·0 and 7·2 for microsomes of Fc and CSMA houseflies respectively. Guidelines were established to reduce errors in measurement and interpretation of substrate difference spectra. Cytochrome P-420 did not exhibit normal type I and type II difference spectra, however, its presence in microsomes interfered with the evaluation of the type III difference spectra of ethyl isocyanide and pyridine. Some strain specific differences were noted in the oxidized difference spectra of type I and type II ligands and in those obtained with microsomes pretreated with either NADH or NADPH. Microsomes from neither strain exhibited normal type I difference spectra with benzphetamine in the presence of dithionite. The data obtained are discussed in relation to current methodology used with microsomal cytochrome P-450 from insects and other taxonomic groups.

Effect of assay conditions on the magnesium requirement of the transfer reaction catalyzed by phenylalanyl-tRNA synthetase from bakers' yeast

Phenylalanyl-tRNA synthetase exhibits an absolute requirement for magnesium ion in its transfer reaction when assayed in 10 mM Tris-acetate buffer at pH 7.2. This magnesium requirement can be largely eliminated by the use of 50 mM sodium cacodylate, citrate or succinate buffers at pH 6. It is thus demonstrated that, depending upon the assay conditions which are employed, an aminoacyl-tRNA synthetase can exhibit ambivalence with respect to the magnesium requirement of its transfer reaction.