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1.
J Vet Diagn Invest ; 32(3): 401-408, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32306865

RESUMO

Neospora caninum is a protozoan parasite that causes abortion and important economic losses in cattle worldwide. There are no treatments or vaccines available; disease control is based on diagnosis and herd management strategies. We developed, validated, and evaluated under field conditions a competitive inhibition ELISA based on the truncated SAG1 protein (tSAG1), expressed in Escherichia coli, and the RafNeo5 monoclonal antibody (ciELISAtSAG1). A criterion based on the 3-y sequential serologic analysis of 230 dairy cows by IFAT was used as the gold standard. The assay was validated using 860 serum samples from cows that were consistently positive or negative by IFAT throughout the study period. ciELISAtSAG1 was then used to evaluate the prevalence of neosporosis in 16 beef cow herds (22 samples per herd, 352 total samples). The results were compared with those from IFAT and a commercial cELISA (cELISAVMRD). The ciELISAtSAG1 cutoff was ≥ 29%I, with a diagnostic sensitivity of 98.7% (95% CI = 96.8-99.7%) and a diagnostic specificity of 97.9% (95% CI = 96.4-99.0%). Concordance among IFAT, cELISAVMRD, and ciELISAtSAG1 was 90.3%. The agreement (κ) between ciELISAtSAG1 and the other 2 tests was ≥ 0.81. The overall prevalence of neosporosis in the 16 beef herds was 30% (range: 5-60%). The ciELISAtSAG1 could be useful for large-scale detection of anti-N. caninum antibodies in cattle and seroepidemiologic investigations, given its appropriate sensitivity and specificity, and the simplicity of production.


Assuntos
Anticorpos Antiprotozoários/sangue , Doenças dos Bovinos/diagnóstico , Coccidiose/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Neospora/isolamento & purificação , Proteínas de Protozoários/análise , Proteínas Recombinantes/análise , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/diagnóstico , Coccidiose/parasitologia , Ensaio de Imunoadsorção Enzimática/métodos
2.
Talanta ; 209: 120563, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892091

RESUMO

In this study is described an on-line titanium dioxide solid-phase extraction capillary electrophoresis-mass spectrometry (TiO2-SPE-CE-MS) method for the analysis of the glycopeptide glycoforms obtained from the tryptic digests of recombinant human erythropoietin (rhEPO). The O126-glycopeptide of rhEPO was used to optimize the methodology given its importance in quality control of biopharmaceuticals and doping analysis. Several aspects that affect the selective retention and elution, peak efficiency and electrophoretic separation of the O126 glycoforms were investigated to maximize detection sensitivity while minimizing non-specific retention of peptides. Under the optimized conditions, the microcartridge lifetime was around 10 analyses and repeatability was acceptable (%RSD values of 9-11% and 6-11% for migration times and peak areas, respectively). The method was linear between 0.5 and 50 mg L-1 and 10-50 mg L-1 for O126 glycoforms containing NeuAc and NeuGc, respectively, and limits of detection (LODs) were up to 100 times lower than by CE-MS. Although optimized for O-glycopeptides, the method proved also successful for preconcentration of N83-glycopeptides, without compromising the separation between glycopeptide glycoforms with different number of sialic acids. Tryptic digests of other glycoproteins (i.e. human apolipoprotein CIII (APO-C3) and bovine alpha-1-acid glycoprotein (bAGP)) were also analyzed, demonstrating the applicability to glycopeptides with different glycan composition and nature.


Assuntos
Eletroforese Capilar/métodos , Eritropoetina/análise , Glicopeptídeos/análise , Extração em Fase Sólida/métodos , Titânio/química , Biomarcadores/análise , Eletroforese Capilar/instrumentação , Desenho de Equipamento , Eritropoetina/isolamento & purificação , Glicopeptídeos/isolamento & purificação , Humanos , Espectrometria de Massas/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/isolamento & purificação , Extração em Fase Sólida/instrumentação
3.
J Chromatogr A ; 1610: 460539, 2020 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-31543341

RESUMO

Over the past decade significant progress has been found in the upstream production processes, shifting the main bottlenecks in current manufacturing platforms for biopharmaceuticals towards the downstream processing. Challenges in the purification process include reducing the production costs, developing robust and efficient purification processes as well as integrating both upstream and downstream processes. Microfluidic technologies have recently emerged as effective tools for expediting bioprocess design in a cost-effective manner, since a large number of variables can be evaluated in a small time frame, using reduced volumes and manpower. Their modularity also allows to integrate different unit operations into a single chip, and consequently to evaluate the effect of each stage on the overall process efficiency. This paper describes the development of a diffusion-based microfluidic device for the rapid screening of continuous chemical lysis conditions. The release of a recombinant green fluorescent protein (GFP) expressed in Escherichia coli (E. coli) was used as model system due to the simple evaluation of cell growth and product concentration by fluorescence. The concept can be further applied to any biopharmaceutical production platform. The microfluidic device was successfully used to test the lytic effect of both enzymatic and chemical lysis solutions, with lysis efficiency of about 60% and close to 100%, respectively, achieved. The microfluidic technology also demonstrated the ability to detect potential process issues, such as the increased viscosity related with the rapid release of genomic material, that can arise for specific lysis conditions and hinder the performance of a bioprocess. Finally, given the continuous operation of the lysis chip, the microfluidic technology has the potential to be integrated with other microfluidic modules in order to model a fully continuous biomanufacturing process on a chip.


Assuntos
Bactérias , Técnicas Analíticas Microfluídicas , Proteínas Recombinantes , Bactérias/química , Bactérias/citologia , Bactérias/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo
5.
Int. microbiol ; 22(4): 471-478, dic. 2019. graf, tab
Artigo em Inglês | IBECS | ID: ibc-185065

RESUMO

Chlamydia trachomatis is considered as a public health problem due to its high prevalence and increased rates of gynecological disorders. The major outer membrane protein (MOMP) of this bacterium is the most abundant protein in its membrane and has been evaluated not only as a vaccine development candidate but also is used in many diagnostic tests. The MOMP weighs 69 kDa and contains four variable segments (VS 1-4) separated by constant regions. Several research groups have developed recombinant single-variable segments of MOMP expressed in Escherichia coli cytoplasm. But, all variable segments have been used minimally for the diagnosis of a chlamydial infection. In this experiment, the authors obtained the recombinant MOMP of C. trachomatis (rMOMP) in E. coli rMOMP and extracted, purified, and partially characterized it. This was later used to identify anti-Chlamydia trachomatis antibodies in sera of infertile patients by immunodetection assays, enzyme-linked immunosorbent assay (ELISA), and indirect immunofluorescence tests. The ELISA test showed high sensitivity and low specificity of 100 and 58.3%, respectively. The above results obtained were linked to the cross-reactivity of antibodies against C. pneumoniae or C. psittaci. Hence, an evaluation was performed to obtain an optimized test for the diagnosis of C. trachomatis infection


No disponible


Assuntos
Antígenos de Bactérias/análise , Chlamydia trachomatis/isolamento & purificação , Infecções por Chlamydia/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Recombinantes/análise , Proteínas da Membrana Bacteriana Externa/metabolismo , Chlamydia trachomatis/genética , Infecções por Chlamydia/microbiologia , Proteínas Recombinantes/genética
6.
Electrophoresis ; 40(23-24): 3084-3091, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31663138

RESUMO

A microfluidic system has been designed that integrates both imaged capillary isoelectric focusing (iCIEF) separations and downstream MS detection into a single assay. Along with the construction of novel instrumentation and an innovative microfluidic chip, conversion to MS-compatible separation reagents has also been established. Incorporation of 280 nm absorbance iCIEF-MS analysis not only permits photometric quantitation of separated charge isoforms but also facilitates the direct monitoring of analyte focusing and mobilization in real-time. The outcome of this effort is a device with the unique ability to allow for both the characterization and identification of protein charge and mass isoforms in under 15 min. Acquisition, quantitation, and identification of highly resolved intact mAb charge isoforms along with their critical N-linked glycan pairs clearly demonstrate analytical utility of our innovative system. In total, 33 separate molecular features were characterized by the iCIEF-MS system representing a dramatic increase in the ability to monitor multiple intact mAb critical quality attributes in a single comprehensive assay. Unlike previously reported CIEF-MS results, relatively high ampholyte concentrations, of up to 4% v/v, were employed without impacting MS sensitivity, observed to be on the order of 1% composition.


Assuntos
Anticorpos Monoclonais/análise , Eletroforese Capilar/métodos , Focalização Isoelétrica/métodos , Espectrometria de Massas/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Anticorpos Monoclonais/química , Medicamentos Biossimilares/análise , Medicamentos Biossimilares/química , Desenho de Equipamento , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Proteínas Recombinantes/análise , Proteínas Recombinantes/química
7.
Lipids ; 54(9): 571-579, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31478204

RESUMO

Phospholipid:diacylglycerol acyltransferase (PDAT) catalyzes the acyl-CoA-independent triacylglycerol (TAG) biosynthesis in plants and oleaginous microorganisms and thus is a key target in lipid research. The conventional in vitro PDAT activity assay involves the use of radiolabeled substrates, which, however, are expensive and demand strict regulation. In this study, a reliable fluorescence-based method using nitrobenzoxadiazole-labeled diacylglycerol (NBD-DAG) as an alternative substrate was established and subsequently used to characterize the enzyme activity and kinetics of a recombinant Arabidopsis thaliana PDAT1 (AtPDAT1). We also demonstrate that the highly toxic benzene used in typical PDAT assays can be substituted with diethyl ether without affecting the formation rate of NBD-TAG. Overall, this method works well with a broad range of PDAT protein content and shows linear correlation with the conventional method with radiolabeled substrates, and thus may be applicable to PDAT from various plant and microorganism species.


Assuntos
Aciltransferases/análise , Arabidopsis/enzimologia , Benzoxazóis/química , Fluorescência , Corantes Fluorescentes/química , Glicerídeos/química , Aciltransferases/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência
8.
Clin Appl Thromb Hemost ; 25: 1076029619873976, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31496264

RESUMO

Patients with von Willebrand disease (VWD) often require treatment with supplemental von Willebrand factor (VWF) prior to procedures or to treat bleeding. Commercial VWF concentrates and more recently recombinant human VWF (rVWF) have replaced cryoprecipitate as the mainstay of therapy. In comparison with cryoprecipitate, the VWF content and multimer distribution under current manufacturing processes of these commercial products has not been reported. We measured the factor VIII (FVIII:C), VWF antigen (VWF:Ag), VWF collagen-binding activity (VWF:CB), VWF platelet-binding activity by GPIbM enzyme-linked immunosorbent assay (VWF:GPIbM), and percentage of high-molecular-weight (HMWM) VWF in 3 pools of group A and O cryoprecipitate, 3 vials of VWF concentrate (Humate-P), and 1 lot of rVWF (Vonvendi). We found that both group O and group A cryoprecipitate have significantly higher ratios of VWF:GPIbM activity and FVIII:C activity relative to VWF:Ag and have better preservation of HMWM than Humate-P. Although not compared statistically, rVWF appears to have more HMWM VWF and a higher ratio of VWF:GPIbM to VWF:Ag than Humate-P and cryoprecipitate. The estimated acquisition cost for our hospital for treating one major bleeding episode was more than 4-fold higher with Humate-P and 7- to 10-fold higher with rVWF than with cryoprecipitate.


Assuntos
Doenças de von Willebrand/tratamento farmacológico , Fator de von Willebrand/análise , Ensaio de Imunoadsorção Enzimática/métodos , Fator VIII/análise , Fibrinogênio/análise , Hemorragia/tratamento farmacológico , Hemorragia/economia , Humanos , Proteínas Recombinantes/análise , Doenças de von Willebrand/economia
9.
Biochim Biophys Acta Mol Cell Res ; 1866(12): 118556, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31505170

RESUMO

Degradation of unwanted proteins is important in protein quality control cooperating with the dynein/dynactin-mediated trafficking along the acetylated microtubule (MT) network. Proteins associated directly/indirectly with tubulin/MTs play crucial roles in both physiological and pathological processes. Our studies focus on the interrelationship of the tubulin deacetylase HDAC6, the MT-associated TPPP/p25 with its deacetylase inhibitory potency and the hub dynein light chain DYNLL/LC8, constituent of dynein and numerous other protein complexes. In this paper, evidence is provided for the direct interaction of DYNLL/LC8 with TPPP/p25 and HDAC6 and their assembly into binary/ternary complexes with functional potency. The in vitro binding data was obtained with recombinant proteins and used for mathematical modelling. These data and visualization of their localizations by bimolecular fluorescence complementation technology and immunofluorescence microscopy in HeLa cells revealed the promoting effect of TPPP/p25 on the interaction of DYNLL/LC8 with both tubulin and HDAC6. Localization of the LC8-2-TPPP/p25 complex was observed on the MT network in contrast to the LC8-2-HDAC6 complex, which was partly translocated to the nucleus. LC8-2 did not influence directly the acetylation of the MT network. However, the binding of TPPP/p25 to a new binding site of DYNLL/LC8, outside the canonical binding groove, counteracted the TPPP/p25-derived hyperacetylation of the MT network. Our data suggest that multiple associations of the regulatory proteins of the MT network could ensure fine tuning in the regulation of the intracellular trafficking process either by the complexation of DYNLL/LC8 with new partners or indirectly by the modulation of the acetylation level of the MT network.


Assuntos
Dineínas do Citoplasma/metabolismo , Desacetilase 6 de Histona/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Dineínas do Citoplasma/análise , Células HeLa , Desacetilase 6 de Histona/análise , Humanos , Proteínas do Tecido Nervoso/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo
10.
Anal Chim Acta ; 1086: 110-115, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31561785

RESUMO

Estrogen receptors (ERs) play a major role in the signaling pathways and participate in regulating and maintaining the basic activities of life. The abnormal expression of ERs has a significant effect on tumorigenesis. Herein, we propose an electrochemical method for detecting ERs based on the formation of DNA Y-Junction with a 3'-blunt end. The DNA Y-junction was designed to have one of its arms bound to an ER. When an ER was bound to the junction, which was immobilized on the electrode surface, it protected the DNA Y-junction from Exo III-catalyzed digestion. DNA Y-Junction also contained G-quadruplex rich duplex, which generated electrochemical signals when hemin was added to the electrode, resulting in the quantitative detection of ERs. The detection range of the ER using this method was 0.1-200 nM with a detection limit of 0.034 nM. Since this assay can be employed to detect ERs in tumor cells, it may be useful in tumor diagnosis in the future.


Assuntos
Técnicas Biossensoriais , DNA/química , Técnicas Eletroquímicas , Receptores Estrogênicos/análise , Sondas de DNA/química , Eletrodos , Humanos , Células MCF-7 , Proteínas Recombinantes/análise , Células Tumorais Cultivadas
11.
Int J Lab Hematol ; 41(5): 679-683, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421012

RESUMO

INTRODUCTION: The measurements of clotting factor activities are usually performed using a one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). Advances in automated coagulation analysers have led to the utilization of stored calibration curves. There are sometimes substantial intervals between test calibration and analysis of samples. Variability in results can be influenced by calibrant and methodology. Several guidelines recommend calibration and patient samples be performed together in parallel; this incurs costs, but reliance on a stored calibration curve may lead to inaccuracy of results over time. METHODS: We evaluated inclusion of a live truncated (3 point) calibration curve using calibrator plasma alongside test samples and compared results calculated against the stored calibration curves and live truncated calibration to assess the impact on precision and accuracy. The feasibility of this was tested on two hospital sites in the UK; OSA and CSA were performed on Sysmex CS5100 using Actin FS, SynthASil, Innovin, Biophen chromogenic VIII and Rossix chromogenic IX. RESULTS: Results of two batches of IQC were compared for FII, FV, FVII, FX, FIX:C, FXI, FXII and OSA FVIII (FVIII:C1) and CSA FVIII:C (FVIII:CR) and FXI:C. By utilizing a live truncated calibration, precision improved with the most striking examples: FXII %CV 23.1% to 3.1% (site A) FXI 7.3% to 2.4% (site B). The improvement in other clotting factors was more modest. CONCLUSION: To the best of our knowledge, this is the first study that demonstrates that the use of a live truncated calibration curve will improve precision and accuracy.


Assuntos
Fatores de Coagulação Sanguínea/análise , Testes de Coagulação Sanguínea/instrumentação , Testes de Coagulação Sanguínea/métodos , Coagulação Sanguínea , Calibragem/normas , Fatores de Coagulação Sanguínea/metabolismo , Testes de Coagulação Sanguínea/economia , Compostos Cromogênicos , Análise Custo-Benefício , Fator VIII/análise , Fator VIII/metabolismo , Humanos , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Padrões de Referência , Reprodutibilidade dos Testes , Tromboplastina/análise , Tromboplastina/metabolismo
12.
Comp Immunol Microbiol Infect Dis ; 66: 101338, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31437683

RESUMO

Three screening tests {(newly developed, six recombinant secretory proteins based 'cocktail ELISA', in-house robust 'indigenous ELISA' based on semi-purified protoplasmic antigens and tissue microscopy were evaluated with 'Gold standard', histo-pathology for the diagnosis of Johne's disease in goats and buffaloes. Serum and tissues {mesenteric lymph nodes and intestines) were driven from farmer's goats (n = 77) and buffaloes (n = 40) slaughtered for harvesting meat and farm goats (n = 77), died and necropsied. Twenty seven (35%) goats and 23 (57.5%) buffaloes were positive in all the four tests. Of 134 tissues screened by histo-pathology, 79.8% MLN and 76.8%, intestines, were positive for MAP infection. In tissue microscopy, 55.2 and 52.3%, goats and buffaloes were positive, respectively. Of 117 sera screened by i_ELISA, 58.4 and 70.0%, goats and buffaloes were positive, respectively. Whereas, c_ELISA detected 55.8 and 62.5%, goats and buffaloes, positives, respectively. Twelve tissues (70.5%) of goats necropsied were positive, both in tissue microscopy and histo-pathology. Most significant gross findings were serous atrophy of the fat and mild to moderate, diffuse thickening of terminal ileum, especially at ileo-caecal junction with or without transverse / longitudinal corrugations. In histo-pathology grade III and IV lesions were significantly low as compared to grade I and II. Of the four tests used for screening 268 samples, histo-pathology was most sensitive (78.3%), followed by i_ELISA (62.3%), c_ELISA (58.9%) and tissue microscopy (58.9%). Between two ELISA tests, c_ELISA using six recombinants secretory proteins, had higher specificity as compared to i_ELISA.


Assuntos
Búfalos/microbiologia , Ensaio de Imunoadsorção Enzimática/métodos , Cabras/microbiologia , Paratuberculose/diagnóstico , Patologia/normas , Proteínas Recombinantes/análise , Matadouros , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/microbiologia , Fezes/microbiologia , Doenças das Cabras/diagnóstico , Doenças das Cabras/microbiologia , Técnicas Histológicas , Microscopia/métodos , Paratuberculose/patologia , Patologia/métodos , Sensibilidade e Especificidade , Coloração e Rotulagem
13.
Biomed Chromatogr ; 33(12): e4686, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31452214

RESUMO

Researchers frequently use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) prior to mass spectrometric analysis in a proteomics approach. The i2D-PAGE method, which 'inverts' the dimension of protein separation of the conventional 2D-PAGE, is presented in this publication. Protein lysate of Channa striata, a freshwater snakehead fish, was separated based on its molecular weight in the first dimension and its isoelectric point in the second dimension. The first-dimension separation was conducted on a gel-free separation device, and the protein mixture was fractionated into 12 fractions in chronological order of increasing molecular weight. The second-dimension separation featured isoelectric focusing, which further separated the proteins within the same fraction according to their respective isoelectric point. Advantages of i2D-PAGE include better visualisation of the isolated protein, easy identification on protein isoforms, shorter running time, customisability and reproducibility. Erythropoietin standard was applied to i2D-PAGE to show its effectiveness for separating protein isoforms. Various staining methods such as Coomassie blue staining and silver staining are also applicable to i2D-PAGE. Overall, the i2D-PAGE separation method effectively separates protein lysate and is suitable for application in proteomics research.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Isoformas de Proteínas/isolamento & purificação , Focalização Isoelétrica/métodos , Peso Molecular , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Proteômica/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Reprodutibilidade dos Testes
14.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1126-1127: 121732, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31376580

RESUMO

A certified reference material (CRM) for the quantification of protein, essential to manage quality control and quality assurance in protein-related works, has been developed. Amino acid analysis with conventional acid hydrolysis and isotope dilution HPLC-MS was used to establish an SI-traceable absolute protein quantification method using recombinant human growth hormone (hGH) as a model protein. The certification method was verified by comparative studies between 1) different methods of protein quantification based on microwave-assisted hydrolysis, and 2) different labs as part of the Asian Collaboration on Reference Material project with Japan, China, and Korea. Certification, evaluation of measurement uncertainty, homogeneity testing, and stability testing were carried out, after which the candidate CRM for hGH quantification was successfully certified with excellent agreement within the certified value in the two comparative studies. Although the quantification value of hGH by amino acid analysis showed good robustness in various conditions, results of intact protein analysis showed degradation profiles in temperatures higher than 4 °C. Consequently, storage and dissemination conditions should be set in accordance with stability tests. Based on the results, this method is believed to be suitable for accurate quantification of hGH. Additionally, it can also be used as a guide to preparation of CRM, and instructions for quality management of protein work for other similar proteins.


Assuntos
Hormônio do Crescimento Humano , Proteínas Recombinantes , Cromatografia Líquida de Alta Pressão/normas , Hormônio do Crescimento Humano/análise , Hormônio do Crescimento Humano/química , Humanos , Espectrometria de Massas/normas , Estabilidade Proteica , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Padrões de Referência
15.
FEBS Open Bio ; 9(10): 1734-1743, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31376210

RESUMO

Lysophosphatidylcholine acyltransferase 3 (LPCAT3) is an important enzyme in phospholipid remodeling, a process that influences the biophysical properties of cell membranes and thus cell function. Multiple lines of evidence suggest that LPCAT3 is involved in several diseases, including atherosclerosis, non-alcoholic steatohepatitis, and carcinoma. Thus, LPCAT3 may have potential as a therapeutic target for these diseases. In the present study, we devised an assay based on reversed-phase HPLC to measure LPCAT3 activity, which may facilitate the identification of LPCAT3 inhibitors and activators. We found that optimal pH and temperature of recombinant human LPCAT3 are 6.0 and 30 °C, respectively. The enzyme Km values for substrates NBD-labelled lysophosphatidylcholine and arachidonoyl CoA were 266.84 ± 3.65 and 11.03 ± 0.51 µmol·L-1 , respectively, and the Vmax was 39.76 ± 1.86 pmol·min-1 ·U-1 . Moreover, we used our new method to determine the IC50 of a known LPCAT inhibitor, TSI-10. In conclusion, this novel assay can be used to measure the effects of compounds on LPCAT3 activity.


Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/análise , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Ensaios Enzimáticos/métodos , 1-Acilglicerofosfocolina O-Aciltransferase/antagonistas & inibidores , Animais , Inibidores Enzimáticos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Temperatura
16.
Bioelectrochemistry ; 130: 107326, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31295699

RESUMO

Diabetes mellitus can be considered one of the most widespread diseases globally. Hence, the diabetes research is currently focused on developing an effective, low-cost sensor having high stability and suitable analytical characteristics. Screen printed carbon electrodes (SPCEs) embody ideal candidates for insulin determination due to the small area of the working electrode eliminating the solution volume required for the given purpose. Modification of SPCEs by using nanoparticles resulted in an increase of the working electrode surface area and formation of a higher number of active species. The aim of this paper is to examine the impact of a chitosan membrane on the electrochemical determination of insulin on NiO nanoparticles (NiONPs) and multi-walled nanotube (MWCNTs) modified SPCE (NiONPs/MWCNTs/SPCE). This study is primarily conceived to compare the analytical characteristics and stability of NiONPs/chitosan-MWCNTs/SPCE and NiONPs/MWCNTs/SPCE. An electrode modified with chitosan displays a wider linear range, one of 0.25 µM - 5 µM (R2 0.997); a lower limit of detection, 94 nM; a high sensitivity (0.021 µA/µM) and better stability than that of an electrode without chitosan. According to these characteristics, the polymer is considered a necessary compound of the electrochemical insulin sensor, improving the sensor's analytical characteristics.


Assuntos
Carbono/química , Quitosana/química , Insulina/análise , Membranas Artificiais , Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Eletrodos , Humanos , Limite de Detecção , Nanopartículas/química , Nanotubos de Carbono/química , Níquel/química , Proteínas Recombinantes/análise
17.
Bioelectrochemistry ; 130: 107322, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31295701

RESUMO

In this work, a selective and simple electrochemical aptasensor was developed for the detection of activated protein C by employing methylene blue (MB) as a redox indicator. An activated protein C aptamer (APC-apt) was covalently immobilized on the surface of a carbon paste electrode modified with gold nanoparticle - chitosan /graphene paste (AuNPs-Chi/Gr). The AuNPs-Chi/Gr paste increased electrochemical peak current and immobilized the aptamer on the electrode surface. The process of aptasensor construction and successful immobilization of the aptamer on the electrode surface was confirmed by electrochemical cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Differential pulse voltammetry (DPV) was used to determine the methylene blue peak current. By replacing APC instead of MB at the electrode surface, the cathodic current of the MB decreases, and this decrease corresponds to the APC concentration. Under optimum conditions, the APC concentration was detected in the range from of 0.1 ng·mL-1 to 40 µg·mL-1 with a relatively low detection limit of 0.073 ng·mL-1. This method was then applied to the determination of APC in human serum samples. The results revealed that this strategy can be used to measure other proteins in biological samples.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Quitosana/química , Grafite/química , Adulto , Carbono/química , Técnicas Eletroquímicas/métodos , Eletrodos , Feminino , Ouro/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Azul de Metileno/química , Pessoa de Meia-Idade , Proteína C/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/sangue
18.
Electrophoresis ; 40(21): 2888-2898, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31271455

RESUMO

Fragmentation in protein-based molecules continues to be a challenge during manufacturing and storage, and requires an appropriate control strategy to ensure purity and integrity of the drug product. Electrophoretic and chromatographic methods are commonly used for monitoring the fragments. However, size-exclusion chromatography often suffers from low resolution of low molecular weight fragments. Electrophoretic methods like CE-SDS are not compatible with enriching fragments for additional characterization tests such as MS. These limitations may result in inadequate control strategy for monitoring and characterizing fragments for protein-based molecules. Capillary western blotting was used in this study as an orthogonal method for characterization of fragments in an IgG1 antibody under reduced conditions. To achieve a comprehensive mapping of various fragments generated by thermal stress, capillary western profiles were generated using recognition antibodies for IgG kappa (κ) light chain, Fc, and Fab regions that enabled unambiguous fragment identification. Additionally, three different enzymatic digestion methods (IdeS, PNGase F, and IgdE) were applied coupled with capillary western blotting for clip identifications. Finally, complementary data collected using traditional chromatographic and electrophoretic methods allowed to establish a comparison of analytical profiles with an added benefit of fragment identification offered by capillary western profiling. In addition to various Fc and Fab-related low molecular weight fragments, a non-reducible thio-ether linked 75 kDa HL fragment was also identified.


Assuntos
Western Blotting/métodos , Eletroforese Capilar/métodos , Fragmentos de Imunoglobulinas , Mapeamento de Peptídeos/métodos , Humanos , Fragmentos de Imunoglobulinas/análise , Fragmentos de Imunoglobulinas/química , Imunoglobulina G/análise , Imunoglobulina G/química , Proteínas Recombinantes/análise , Proteínas Recombinantes/química
19.
Biotechnol J ; 14(11): e1800556, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31329337

RESUMO

The performance of a bioreactor in meeting process goals is affected by the microorganism used, medium composition, and operating conditions. A typical bioreactor uses a supervisory control and data acquisition (SCADA) system for control, and a combination of software and hardware tools for real-time data analysis. However, when the process is disrupted by utility or instrumentation failure, typical process controllers may be unable to reinstate normal operating conditions before the cells in the reactor shift to unfavorable metabolic regimes. The objective of this study is to examine how the response of a controller affects process recovery when disruptive incidences occur under a process analytical technology (PAT) framework. The process used for this investigation is the production of lethal toxin-neutralizing factor (LTNF) by Escherichia coli (E. coli), which is controlled by a decoupled input-output-linearizing controller (DIOLC). The performance of the DIOLC is compared to a proportional integral derivative (PID) controller subjected to the same conditions. The disruptions are introduced manually and the effect of controller action on process recovery and LTNF synthesis is measured in terms of peak purity and concentration. It is observed that DIOLC performs better after reinstating operating conditions and results in a meaningful improvement in performance.


Assuntos
Escherichia coli/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/análise , Reatores Biológicos , Meios de Cultura , Fermentação , Cinética , Modelos Biológicos , Software
20.
Virology ; 535: 171-178, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31306912

RESUMO

Human respiratory syncytial virus (RSV) is one of the predominant pathogens causing lower respiratory tract infection in infants and young children worldwide, whereas there is so far no vaccine or drug against RSV infection for clinical use. In this work, we developed and validated a fluorescence-based high-throughput screening (HTS) assay to identify compounds active against RSV, using RSV-mGFP, a recombinant RSV encoding enhanced green fluorescent protein (EGFP). Thereafter, among 54,800 compounds used for our screen, we obtained 62 compounds active against RSV. Among these hits, azathioprine (AZA) and 6-mercaptopurine (6-MP) were identified as RSV inhibitors with half maximal inhibitory concentration (IC50) values of 6.69 ±â€¯1.41 and 3.13 ±â€¯0.98 µM, respectively. Further experiments revealed that they functioned by targeting virus transcription or/and genome replication. In conclusion, the established HTS assay is suitable to screen anti-RSV compounds, and the screened two hits of AZA and 6-MP, as potential anti-RSV agents targeting RSV genome replication/transcription, are worthy of further investigation on their anti-RSV activity in vivo.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Coloração e Rotulagem/métodos
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