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1.
Chemosphere ; 251: 126626, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32443247

RESUMO

Three spectrophotometric methods have been developed and compared for the quantification of low concentrations (0.03-63 µM) of aqueous permanganate in neutral pH conditions. Although permanganate is a widely used oxidant in drinking water and wastewater treatment, no widely accepted method of quantification has been reported to date. While one method presented does not require the need for any reagent chemicals (direct spectrophotometric analysis), it yielded a relatively low molar absorption coefficient of 3340 M-1 cm-1 at 525 nm and a level of detection (LOD) and quantification (LOQ) of 0.45 and 1.51 µM, respectively. Some instability of permanganate species during direct quantification was found to occur over 60 min, with a total decrease of 0.002 (arbitrary units) of absorbance, equivalent to a decrease in concentration of 0.6 µM. Beyond 60 min, no further degradation was observed. Indirect spectrophotometric analyses using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and sodium iodide (NaI) provided a significantly more sensitive method for permanganate quantification, yielding molar absorption coefficients of 140,030 and 61,130 M-1 cm-1, respectively. The LOD and LOQ were determined to be 0.01 and 0.03 µM for the ABTS method and 0.02 and 0.08 µM for the NaI method, respectively. Although conservative and accurate limits of quantification for both the ABTS and NaI methods are presented, which should be sufficient of most practical applications, lower limits may be possible with further refinement of the methods.


Assuntos
Compostos de Manganês/análise , Óxidos/análise , Espectrofotometria/métodos , Águas Residuárias/química , Poluentes Químicos da Água/análise , Purificação da Água/métodos , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Limite de Detecção , Padrões de Referência , Espectrofotometria/instrumentação
2.
J Chromatogr A ; 1622: 461100, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32359780

RESUMO

The aim of the present investigation was application of hydrophilic interaction liquid chromatography as an alternative chromatographic approach for the study of antisense oligonucleotides. The influence of several mobile phases, differing with the salt type, their concentration and pH value on the retention and the separation of antisense oligonucleotides has been examined for this purpose. Four different stationary phases were also applied including unmodified silica, silica modified with the use of sulfobetaine groups, polyhydroxy and aminopropyl groups. Such wide range of tested conditions has been useful in better understanding of the retention mechanism of tested compounds. The results obtained during this investigation indicated that greater retention, greater peaks symmetry, as well as more effective separation of oligonucleotides, were obtained for the zwitterionic stationary phase. Moreover, the optimization of tandem mass spectrometry parameters with the use of Central Composite Design was performed and different mobile phases were tested to choose that one, which provided the greatest antisense oligonucleotides peak areas in Multiple Reaction Monitoring mode and consequently, the greatest possible sensitivity. Hydrophilic interaction liquid chromatography was compared with the ion pair chromatography, commonly used in the analysis of oligonucleotides. Both techniques were compared in terms of selectivity of separation as well as the sensitivity of their determination. Obtained results proved that ion pair chromatography provided better results in terms of separation efficiency and peak areas in Multiple Reaction Monitoring for tested conditions. However, these results do not preclude application of hydrophilic interaction liquid chromatography as an alternative chromatographic approach for the oligonucleotides analysis especially when a mobile phase without ion pair reagents is required.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia Líquida , Interações Hidrofóbicas e Hidrofílicas , Oligonucleotídeos Antissenso , Espectrometria de Massas em Tandem , Betaína/análogos & derivados , Betaína/química , Indicadores e Reagentes , Oligonucleotídeos Antissenso/isolamento & purificação , Oligonucleotídeos Antissenso/metabolismo , Dióxido de Silício/química
3.
Zhongguo Yi Liao Qi Xie Za Zhi ; 44(1): 88-91, 2020 Jan 08.
Artigo em Chinês | MEDLINE | ID: mdl-32343076

RESUMO

By analyzing the main problems existing in the current management of medical devices for clinical trials, this study proposes a feasible management model and specific requirements for acceptance, distribution, storage and recovery combining with the characteristics of medical consumable equipment and diagnostic reagent, which provides a favorable guarantee for the authenticity and reliability of clinical trials.


Assuntos
Ensaios Clínicos como Assunto , Equipamentos e Provisões/normas , Indicadores e Reagentes/normas , Projetos de Pesquisa/normas , Reprodutibilidade dos Testes
4.
J Chromatogr A ; 1620: 461012, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32276856

RESUMO

Quantification of analysis results for the suspect and non-targeted screening is essential for obtaining meaningful insight from the measurements. Ionization efficiency predictions is a possible approach to enable quantitation without standard substances. This is, however, especially challenging for the analysis carried out by combining the full scan mode either with fragmentation experiments in data-dependent or data-independent acquisition mode. Here we investigate the correlation of ionization efficiency values measured in full scan mode with the response factors measured in multiple reaction monitoring (MRM) mode for derivatized amino acids. We observe good correlation (R2 of 0.80) for 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized amino acids. This encourages the use of the measured ionization efficiency values to estimate amino acid concentrations in different beverages. We apply the measured ionization efficiency values for estimating the concentration of amino acids for measurements done both in full scan as well as in MRM mode in wines and beers. We show that the calculated concentrations are in very good correlation with measured values (R2 of 0.71 to 1.00). The method possesses average trueness of 70.5% and shows an insignificant matrix effect.


Assuntos
Aminoácidos/análise , Cromatografia Líquida/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Vinho/análise , Aminas/análise , Aminoácidos/química , Aminoquinolinas/química , Cerveja/análise , Carbamatos/química , Indicadores e Reagentes , Malonatos/química , Reprodutibilidade dos Testes
5.
Klin Lab Diagn ; 65(2): 106-110, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32159308

RESUMO

Currently, the WHO laboratory manual for the examination and processing of human semen (5th edition, 2010) provides updated, standardized, evidence-based procedures and recommendations for laboratory managers, scientists and technicians to follow in examining human semen in a clinical or research setting. Despite the fact, there are several gaps and limitations in the interpretation of this compendium. Mostly, the WHO-protocol of estimation of peroxidase-positive cells and spermatozoa, as well as evaluation of their viability and morphology are not so affordable and applicable in Russia due to peculiarities of laboratory market. Furthermore, most of Russian manuscripts do not reflect a unified approach to the analytical stage of semen analyses. In order to standardize the protocol for human semen examination which adopted to Russian lab it was developed packing of reagents (GEMSTANDART-SEMEN ANALYSES, LLC 'GEMSTANDART', Saint Petersburg, Russia) allowing to obtain an accuracy and completeness of the examination. In summary, this approach is a necessary step in male fertility evaluation. Together with a clinical information, it is indispensable for planning the appropriate clinical management and tacking care in male health.


Assuntos
Análise do Sêmen/normas , Sêmen , Humanos , Indicadores e Reagentes , Masculino , Federação Russa , Espermatozoides
6.
Science ; 367(6482): 1151-1156, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32139547

RESUMO

The regulation of messenger RNA levels in mammalian cells can be achieved by the modulation of synthesis and degradation rates. Metabolic RNA-labeling experiments in bulk have quantified these rates using relatively homogeneous cell populations. However, to determine these rates during complex dynamical processes, for instance during cellular differentiation, single-cell resolution is required. Therefore, we developed a method that simultaneously quantifies metabolically labeled and preexisting unlabeled transcripts in thousands of individual cells. We determined synthesis and degradation rates during the cell cycle and during differentiation of intestinal stem cells, revealing major regulatory strategies. These strategies have distinct consequences for controlling the dynamic range and precision of gene expression. These findings advance our understanding of how individual cells in heterogeneous populations shape their gene expression dynamics.


Assuntos
Estabilidade de RNA , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcrição Genética , Animais , Humanos , Indicadores e Reagentes/química , Células K562 , Camundongos , Uridina/análogos & derivados
7.
Nature ; 579(7799): 379-384, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32188949

RESUMO

Automated synthesis platforms accelerate and simplify the preparation of molecules by removing the physical barriers to organic synthesis. This provides unrestricted access to biopolymers and small molecules via reproducible and directly comparable chemical processes. Current automated multistep syntheses rely on either iterative1-4 or linear processes5-9, and require compromises in terms of versatility and the use of equipment. Here we report an approach towards the automated synthesis of small molecules, based on a series of continuous flow modules that are radially arranged around a central switching station. Using this approach, concise volumes can be exposed to any reaction conditions required for a desired transformation. Sequential, non-simultaneous reactions can be combined to perform multistep processes, enabling the use of variable flow rates, reuse of reactors under different conditions, and the storage of intermediates. This fully automated instrument is capable of both linear and convergent syntheses and does not require manual reconfiguration between different processes. The capabilities of this approach are demonstrated by performing optimizations and multistep syntheses of targets, varying concentrations via inline dilutions, exploring several strategies for the multistep synthesis of the anticonvulsant drug rufinamide10, synthesizing eighteen compounds of two derivative libraries that are prepared using different reaction pathways and chemistries, and using the same reagents to perform metallaphotoredox carbon-nitrogen cross-couplings11 in a photochemical module-all without instrument reconfiguration.


Assuntos
Técnicas de Química Sintética/instrumentação , Técnicas de Química Sintética/métodos , Triazóis/síntese química , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Automação/instrumentação , Automação/métodos , Carbono/química , Indicadores e Reagentes/química , Nitrogênio/química , Oxirredução , Processos Fotoquímicos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Software , Soluções/química , Triazóis/química
8.
PLoS One ; 15(3): e0226467, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32203515

RESUMO

The aim of this study was to compare the Candida bromcresol green (BCG) medium with the chromogenic (CHROM) Brilliance Candida agar and Sabouraud dextrose agar (SDA) media in regard to their capability of detecting Candida isolates from mono- or dual-species cultures. We prepared Candida isolates' suspensions to obtain mono-species (n = 18) or dual-species (n = 153) culture plates per each medium, and three readers independently observed 513 plates at 24-h, 48-h and 72-h incubation time. We scored reading results as correct, over or under detection compared to the expected species number(s). BCG showed significantly higher correct-detection and lower under-detection rates for all Candida species when observed by at least one reader. At 24-h reading, 12 mono-species cultures had correct (or over) detections in all media, whereas 106, 60 and 78 dual-species cultures had correct (or over) detections in BCG, CHROM or SDA, respectively. BCG provides the basis for an accurate laboratory diagnosis of Candida infections.


Assuntos
Ágar/química , Candida/isolamento & purificação , Candidíase/diagnóstico , Meios de Cultura/química , Indicadores e Reagentes/química , Candidíase/microbiologia , Humanos , Técnicas Microbiológicas/métodos
9.
Clin Chim Acta ; 505: 119-124, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32113814

RESUMO

BACKGROUND: Lipoprotein(a) [Lp(a)] is an important cardiovascular risk factor, but clinical immunoassays are flawed. Apolipoprotein(a) [apo(a)], the characteristic protein of Lp(a), contains a variable number of kringle repeats (size isoforms) that make accurate measurement of Lp(a) difficult. We developed a sandwich enzyme immunoassay that uses a murine monoclonal anti-apo(a) antibody for capture and a polyclonal anti-apolipoprotein B (apo B) for detection. Because Lp(a) contains one molecule each of apo(a) and apo B, the assay measures the number of Lp(a) particles [Lp(a)-P] in the circulation without bias due to apo(a) size isoforms. METHODS: After developing and choosing the best anti-apo(a) clone for Lp(a) capture, we identified suitable reagents and ELISA conditions, and validated assay performance (precision, linearity, limit of detection, interferences, and apo(a) size isoform bias). RESULTS: The Lp(a)-P assay was precise with within-run precision of 5.5% to 7.2% and total imprecision of 6.9% to 12.1%. The assay had a limit of detection of 13 nmol/l and was linear from 2 to 499 nmol/l. There was no interference from plasminogen or apolipoprotein B up to 80 and 200 mg/dl, respectively, and bias plot showed no bias related to apo(a) size (kringle 4 type 2 repeats). CONCLUSIONS: Lp(a)-P assay is sensitive, precise and linear over a wide analytical range and is a suitable alternative for laboratories concerned about inaccuracy due to apo(a) size polymorphism and the poor performance of immunoturbidimetric assays.


Assuntos
Apolipoproteínas A/análise , Lipoproteína(a)/análise , Animais , Anticorpos Monoclonais/química , Doenças Cardiovasculares/sangue , Ensaio de Imunoadsorção Enzimática , Humanos , Indicadores e Reagentes , Limite de Detecção , Camundongos , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Isoformas de Proteínas , Reprodutibilidade dos Testes
10.
J Chromatogr A ; 1620: 460983, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32098683

RESUMO

In general counter-current chromatography systems, there are several off-column fittings between injector and column inlet, such as bends, valves, connecting tubes and joints. Due to these off-column fittings, the sample will diffuse in the mobile phase and form an irregular distribution when it flows from the injector to the column inlet. Thus, the concentration distribution of the solutes at the column inlet is a continuous curve (called the injection profile). As some previous research reveals, it is necessary to input actual injection profile into the simulation model to mimic elution profile. Therefore, we built a non-ideal CCC model whose initial value is from the actual injection profile, and validated the rationality of this model with iteration method. The simulation analysis of different injection profiles shows the conditions whereby a discrete injection profile can replace the actual injection profile in the non-ideal CCC model for accurate simulation elution. Simulation elution under such conditions reveal that non-ideal injection model can reflect the relationship between the injection profile and elution profile, and help to explain the reasons of irregular change in elution profile, like the tailed peak and flat peak.


Assuntos
Distribuição Contracorrente/métodos , Modelos Teóricos , Simulação por Computador , Indicadores e Reagentes , Soluções
11.
Nucleic Acids Res ; 48(7): e38, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32064511

RESUMO

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Edição de Genes , Reparo de DNA por Recombinação , Animais , Proteína 9 Associada à CRISPR/genética , Corantes Fluorescentes , Proteínas de Fluorescência Verde/genética , Mutação INDEL , Indicadores e Reagentes , Melanócitos , Nitrorredutases/genética , RNA/química , Moldes Genéticos , Peixe-Zebra/embriologia , Peixe-Zebra/genética
12.
J Chromatogr A ; 1619: 460951, 2020 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-32085914

RESUMO

The mixed-mode chromatographic behavior was estimated for imidazoline and serotonin receptor ligands, and their related compounds on dual hydrophilic/reversed phase stationary phase. The Box-Cox transformation was used to obtain the most suitable mathematical equations which describe the mixed-mode retention. Optimal equations were found for the optimization parameter (λ): λ = -1, λ = -0.5, λ = 0, λ = 0.5, and λ = 1. The proposed equations show satisfactory characteristics compared to standard multimodal and quadratic approaches. For a wide range of volume fractions of the mobile phase modifier, crossing between hydrophilic and reversed phase interactions (the turning point) was defined in terms of the minimal retention and the minimum value of the volume fraction of the aqueous eluent in the mobile phase. The cubic spline interpolation was used as a reference method for estimation of the turning point. It was found out that the newly proposed equations can be used as alternative mathematical forms for the description of the dual retention mechanism and for the evaluation of the turning point. Three new experimental descriptors of the mixed-mode retention were proposed. Two descriptors quantitatively characterize hydrophilic (log kH) and reversed phase (log kR) interactions, while the third one (log kA) refers to the average retention for the whole HILIC/RP range. It was established that the main factors which control dual nature of the mixed-mode retention are lipophilicity, dipol-dipol, van der Waals and hydrogen bonding interactions. It was concluded that the newly proposed estimations of the retention data reliably characterize the mixed-mode chromatographic behavior.


Assuntos
Cromatografia de Fase Reversa , Modelos Teóricos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Imidazolinas , Indicadores e Reagentes , Ligantes
13.
Anal Bioanal Chem ; 412(8): 1741-1755, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32043203

RESUMO

Previously, we demonstrated capture and concentration of Salmonella enterica subspecies enterica ser. Typhimurium using magnetic ionic liquids (MILs), followed by rapid isothermal detection of captured cells via recombinase polymerase amplification (RPA). Here, we report work intended to explore the broader potential of MILs as novel pre-analytical capture reagents in food safety and related applications. Specifically, we evaluated the capacity of the ([P66614+][Ni(hfacac)3-]) ("Ni(II)") MIL to bind a wider range of human pathogens using a panel of Salmonella and Escherichia coli O157:H7 isolates, including a "deep rough" strain of S. Minnesota. We extended this exploration further to include other members of the family Enterobacteriaceae of food safety and clinical or agricultural significance. Both the Ni(II) MIL and the ([P66614+][Dy(hfacac)4-]) ("Dy(III)") MIL were evaluated for their effects on cell viability and structure-function relationships behind observed antimicrobial activities of the Dy(III) MIL were determined. Next, we used flow imaging microscopy (FIM) of Ni(II) MIL dispersions made in model liquid media to examine the impact of increasing ionic complexity on MIL droplet properties as a first step towards understanding the impact of suspension medium properties on MIL dispersion behavior. Finally, we used FIM to examine interactions between the Ni(II) MIL and Serratia marcescens, providing insights into how the MIL may act to capture and concentrate Gram-negative bacteria in aqueous samples, including food suspensions. Together, our results provide further characterization of bacteria-MIL interactions and support the broader utility of the Ni(II) MIL as a cell-friendly capture reagent for sample preparation prior to cultural or molecular analyses. Graphical abstract.


Assuntos
Enterobacteriaceae/metabolismo , Líquidos Iônicos/metabolismo , Magnetismo , Enterobacteriaceae/classificação , Enterobacteriaceae/isolamento & purificação , Indicadores e Reagentes/química , Especificidade da Espécie , Água
14.
Chem Commun (Camb) ; 56(19): 2897-2900, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-32037418

RESUMO

[Tm(DPA)3]3- was used to generate multiple, paramagnetic nuclear Overhauser effect NMR spectra of cationic peptides when weakly bound to a lipopolysaccharide micelle. Increased spectral resolution combined with a marked increase in the number of distance restraints yielded high resolution structures of polymyxin and MSI-594 in the liposaccharide bound state.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Elementos da Série dos Lantanídeos/química , Micelas , Ressonância Magnética Nuclear Biomolecular/métodos , Indicadores e Reagentes/química , Peptídeos/química , Polimixina B/química , Conformação Proteica
15.
Chem Commun (Camb) ; 56(19): 2917-2920, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-32037436

RESUMO

Combinatorial cyclization of hundreds to thousands of random linear peptides by structurally diverse chemical linkers offers access to large macrocyclic compound libraries. A bottleneck in the development of such libraries is the preparation of large numbers of short random linear peptides. Herein, we present a tag-based strategy that is not dependent on a throughput-limiting chromatographic purification step and thus enables parallel production of short peptides. In brief, peptides are synthesized on solid phase as conjugates with a disulfide-linked Cys-Gly-Arg-Trp tetra-peptide tag. The charged arginine residue in the tag allows for purification of the peptides by diethyl ether-precipitation and the tryptophan allows for quantification of the product by absorption measurement. Addition of a reducing agent releases the short peptides from the tag. The released sulfhydryl group in the peptide can readily be used for cyclization of the peptide library with bis-electrophilic linker reagents.


Assuntos
Dissulfetos/química , Oligopeptídeos/química , Cromatografia Líquida , Indicadores e Reagentes/química , Espectrometria de Massas , Oligopeptídeos/síntese química , Oligopeptídeos/isolamento & purificação , Biblioteca de Peptídeos
16.
Nat Commun ; 11(1): 1015, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081914

RESUMO

Many reagents have been developed for cysteine-specific protein modification. However, few of them allow for multi-functionalization of a single Cys residue and disulfide bridging bioconjugation. Herein, we report 3-bromo-5-methylene pyrrolones (3Br-5MPs) as a simple, robust, and versatile class of reagents for cysteine-specific protein modification. These compounds can be facilely synthesized via a one-pot mild reaction and they show comparable tagging efficiency but higher cysteine specificity than the maleimide counterparts. The addition of cysteine to 3Br-5MPs generates conjugates that are amenable to secondary addition by another thiol or cysteine, making 3Br-5MPs valuable for multi-functionalization of a single cysteine and disulfide bridging bioconjugation. The labeling reaction and subsequent treatments are mild enough to produce stable and active protein conjugates for biological applications.


Assuntos
Cisteína/química , Proteínas/química , Técnicas de Química Sintética/métodos , Dissulfetos/química , Indicadores e Reagentes/química , Fenômenos de Química Orgânica , Pirróis/química , Somatostatina/química
17.
Clin Chim Acta ; 505: 98-99, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32035850

RESUMO

BACKGROUND: The fifth generation (high-sensitivity) troponin T assay offers increased precision and analytical sensitivity to the predecessor method. The assay has proven utility in risk stratification and patient management. Upon clinical suspicion and discordant 4th generation troponin T and troponin I results, we investigated a sample for suspected interfering substances to the 5th generation troponin T assay. METHODS: The analysis included a serial dilution, treatment with polyethylene glycol, commercial antibody blocking reagents, and size exclusion chromatography. RESULTS: The sample diluted linearly (R2 = 0.9957); however, experienced a dramatic reduction in concentration after both the polyethylene glycol and blocking agent treatment. Finally, size exclusion chromatography demonstrated assay reactivity around 970 kDa range. CONCLUSIONS: These experiments elucidate a heterophilic antibody interference to the assay, and demonstrate potential measures to discern the interference.


Assuntos
Anticorpos/análise , Troponina T/análise , Idoso , Cromatografia em Gel , Eletrocardiografia , Reações Falso-Positivas , Feminino , Humanos , Imunoensaio , Técnicas de Diluição do Indicador , Indicadores e Reagentes , Imagem por Ressonância Magnética , Polietilenoglicóis/análise , Sensibilidade e Especificidade , Cardiomiopatia de Takotsubo/diagnóstico , Cardiomiopatia de Takotsubo/diagnóstico por imagem
18.
Clin Chim Acta ; 505: 130-135, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32084383

RESUMO

BACKGROUND: Biotin is an interference in many streptavidin-biotin based immunoassays, causing falsely decreased results with sandwich immunoassays and falsely increased results with competitive immunoassays. It has been discussed that premixing streptavidin coated beads and biotinylated capturing molecules may prevent biotin interference. This study was designed to test whether such modification could mitigate biotin interference in two originally susceptible sandwich immunoassays. METHODS: Roche C-peptide and human growth hormone (hGH) immunoassays utilize three reagent containers for streptavidin coated beads (M), biotinylated capturing antibody (R1) and ruthenylated antibody (R2). The reagents were modified by premixing reagent M and R1. Following incubation, the beads were placed back in the M-container and R1-supernatant back to R1-container. Patient specimens were selected, spiked with biotin to 1055 ng/mL, and measured by both the original, unmodified reagent and modified reagent on Roche cobas e411 analyzer. The biotin interference dose response curves were also compared using pooled patient specimen spiked with different concentrations of biotin. RESULTS: For the original reagent, 1055 ng/mL of biotin decreased C- peptide results by 88% and hGH results by 97%. After reagent modification, this interference effect was nearly eliminated for C- peptide but remained about 20% decreased for hGH. CONCLUSION: Premixing streptavidin beads and biotinylated capturing molecules is an effective approach to mitigate biotin interference for certain immunoassays.


Assuntos
Biotina/análise , Biotina/química , Imunoensaio/métodos , Estreptavidina/química , Biotinilação , Peptídeo C/análise , Reações Falso-Positivas , Hormônio do Crescimento Humano/análise , Humanos , Indicadores e Reagentes
19.
Clin Chim Acta ; 505: 73-77, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32092319

RESUMO

BACKGROUND: The objective of the study was to investigate the effectiveness of screening for hereditary galactosaemia with Benedict's test and thin layer chromatography (TLC) in a tertiary laboratory from a developing country. METHODS: We retrospectively analysed the results of tests done in suspected galactosaemia patients including Benedict's test, thin layer chromatography, GALT activity and DNA analysis. RESULTS: 878 paediatric patients were screened with Benedict's test; the age range was 5 days to 19 years. 48% tested positive/trace on the Benedict's test of which 52% of these had galactosuria evident on TLC. 22% of this sample had pathologically low GALT results on follow-up. 8 patients from the screened population were confirmed to have galactosaemia, in addition to 6 more patients diagnosed with galactosaemia without screening tests performed. Median ages at which the diagnoses were made in the screened and non-screened samples were 2 months and 6 months respectively. Confirmatory DNA testing was performed in 2 patients, whom were found to be heterozygous for S135L mutation. CONCLUSION: Inadequate performance of Benedict's test and TLC was demonstrated by false positive and false negative results leading us to conclude that screening test results require interpretation with caution.


Assuntos
Galactosemias/diagnóstico , Programas de Rastreamento/métodos , Adolescente , Criança , Pré-Escolar , Cromatografia em Camada Delgada , Sulfato de Cobre , DNA/genética , Análise Mutacional de DNA , Países em Desenvolvimento , Reações Falso-Negativas , Reações Falso-Positivas , Feminino , Galactosemias/genética , Galactosemias/urina , Humanos , Indicadores e Reagentes , Lactente , Masculino , Reprodutibilidade dos Testes , Estudos Retrospectivos , África do Sul , UTP-Hexose-1-Fosfato Uridililtransferase/análise , UTP-Hexose-1-Fosfato Uridililtransferase/genética , Adulto Jovem
20.
Water Res ; 173: 115521, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32018173

RESUMO

In-vitro diagnostic assays involve various substances that may be released into the environment via a sewage treatment plant if the effluent from diagnostic instruments is discharged into the drains. Because the release of liquid waste into the public sewer system is regulated at national and/or local levels, a risk assessment per site should be performed to determine whether the release of the effluent from in-vitro diagnostic equipment is compliant with applicable regulations. To facilitate the assessment, we developed a screening tool to generate exposure scenarios for chemical substances used in in-vitro diagnostic assays. This screening tool helps in determining a) whether release of the effluent into the sewer system would be allowed from a regulatory point of view and b) whether such release would result in potential risks to surface water. The screening tool is based on conditions encountered at typical usage sites in Switzerland and Germany, with the option to adapt them to other local settings. In addition, measured analytical results of liquid waste together with typical operational parameters of diagnostic equipment were defined as default input parameters. The resulting concentrations in liquid waste and predicted environmental concentrations (PEC) are compared with regulatory limit values for release into the sewer system and predicted no-effect concentrations (PNEC) for surface water for risk estimation. The functioning of the presented screening tool is demonstrated by two assessments performed for fictitious but typical hospitals in Switzerland and Germany. It identified substances that cause potential concern for which a more advanced assessment is required.


Assuntos
Poluentes Químicos da Água , Exposição Ambiental , Monitoramento Ambiental , Alemanha , Indicadores e Reagentes , Medição de Risco , Esgotos , Suíça , Eliminação de Resíduos Líquidos
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