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1.
Artigo em Inglês | MEDLINE | ID: mdl-32130358

RESUMO

Hepatitis B is a severe public health problem. The main world health centers have discussed it due to its pandemic proportion, high pathogenicity and infectivity. This study aimed to determine the immunization profile of dental surgeons - against hepatitis B virus (HBV) in the Public Health System (PHS) through the detection of anti-HBsAg antibodies by immunochromatography and associations with dental surgeons' social and educational profile, factors related to professional knowledge and practices. This is a cross-sectional study on the prevalence of vaccination and immune status to HBV in dentists of Sao Paulo State PHS. Data collection occurred in three stages: questionnaire application, analysis of adhesion to the vaccination protocol and anti-HBsAg tests. Statistical analysis used the Bivariate Analysis and the Binary Logistic Regression. From the total of 219 interviewees, 74.9% reported having received three doses of the vaccine, but 35.6% were not immune to HBV. The dependent variable was associated with years in the public service (years) (OR [Odds Ratio]=1.04; 95% CI 1.00-1.08); knowledge on the subject (OR=6.93; 95% CI 1.39-34.40); incorrect answer concerning the etiological agent of the disease (OR=2.60; 95% CI 1.30-5.22); ignorance on the number of vaccine doses that have been administered (OR=3.43; 95% CI 1.14-10.30); and less than three doses of the vaccine in the immunization schedule (OR=8.76; 95% CI 3.50-21.91). A considerable portion of professionals non-immune to the HBV were found. We concluded that knowledge, years of practice and completion of the vaccination schedule (three doses) affected dental surgeons' immune status to HBV.


Assuntos
Odontólogos/estatística & dados numéricos , Vacinas contra Hepatite B/administração & dosagem , Hepatite B/prevenção & controle , Doenças Profissionais/prevenção & controle , Exposição Ocupacional , Adulto , Cromatografia de Afinidade , Competência Clínica , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Saúde Pública , Fatores Socioeconômicos , Inquéritos e Questionários , Adulto Jovem
3.
Artigo em Inglês | MEDLINE | ID: mdl-32086145

RESUMO

The aim of this study was to develop a rapid and sensitive immunochromatographic test system for the detection of lincomycin (LIN), which belongs to the lincosamide group of antibiotics and contaminates food products of animal origin. Two formats of immunochromatographic analysis (ICA) based on different approaches of introducing gold nanoparticles (GNPs) as a label were compared. It was demonstrated that an indirect ICA method where GNPs were conjugated with anti-species antibodies allowed the achievement of both instrumental and visual detection limits of LIN almost two orders of magnitude lower than those achieved in the standard direct ICA format. In the optimized conditions, the developed indirect ICA allowed for the detection of LIN within 15 min, with instrumental and visual detection limits of 8 pg/mL and 0.8 ng/mL. The assay showed 40% cross-reactivity to clindamycin (CLIN) as a structural analogue of LIN, with no interaction with antibiotics from other classes. The developed ICA was applied for LIN detection in a panel of food products. No treatment of cow milk was necessary before the analysis. For chicken eggs and honey, a simple procedure of preliminary sample preparation was developed, which fully prevented a matrix influence on the assay results. It was demonstrated that ICA could detect LIN in food products while preserving the same analytical characteristics as in the buffer. The analytical recoveries of LIN in foodstuffs were 93.8-125% with coefficients of variations of 5.3-14.0%.


Assuntos
Cromatografia de Afinidade/métodos , Resíduos de Drogas/análise , Lincomicina/análise , Animais , Bovinos , Resíduos de Drogas/isolamento & purificação , Limite de Detecção , Lincomicina/isolamento & purificação , Leite/química
4.
Anal Bioanal Chem ; 412(4): 895-904, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31900533

RESUMO

We have developed an aptamer affinity column (AAC) for the purification and enrichment of trace aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) in genuine agro-products through the covalent conjugation of amino modified aptamer and NHS-activated Sepharose. The coupling and working conditions found to be suitable for this AFB-AAC were examined in regard to coupling time (2 min), loading volume (30 mL), and the methanol concentration (< 10%) used in the loading step. The performance of AFB-AAC was then further evaluated in terms of capacity (329.1 ± 13.7 ng for AFB1 and 162.5 ± 8.9 ng for AFB2), selectivity (excellent), reusability (twenty-three times for AFB1 and twelve times for AFB2), and repeatability (92.7% ± 2.9% for AFB1 and 71.5% ± 3.4% for AFB2). Furthermore, the AAC clean-up combined with HPLC-FLD demonstrated excellent linearity over a wide range, good sensitivity with an LOD of 50 pg mL-1 for AFB1 and 15 pg mL-1 for AFB2, and acceptable recovery with different spiking levels in different matrices. Finally, the AAC was successfully applied to analyte AFB1 and AFB2 in four types of agro-products as well as a maize flour reference material, and the results were found to be in accordance with those of commercial IACs. This study provides a reference for the analysis of other trace analytes by merely changing the corresponding aptamer and represents a strong contender for immune affinity columns. Graphical abstract An aptamer affinity column for purification and enrichment of aflatoxin B1 and aflatoxin B2 in agro-products with the aid of HPLC-FLD and a post-column photochemical derivatization reactor.


Assuntos
Aflatoxina B1/isolamento & purificação , Aflatoxinas/isolamento & purificação , Aptâmeros de Nucleotídeos/química , Cromatografia de Afinidade/métodos , Aflatoxina B1/análise , Aflatoxinas/análise , Arachis/química , Cromatografia Líquida de Alta Pressão/métodos , Análise de Alimentos/métodos , Limite de Detecção , Oryza/química , Triticum/química , Zea mays/química
5.
PLoS One ; 15(1): e0220095, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910206

RESUMO

There are numerous reports on poly-ß-hydroxybutyrate (PHB) depolymerases produced by various microorganisms isolated from various habitats, however, reports on PHB depolymerase production by an isolate from plastic rich sites scares. Although PHB has attracted commercial significance, the inefficient production and recovery methods, inefficient purification of PHB depolymerase and lack of ample knowledge on PHB degradation by PHB depolymerase have hampered its large scale commercialization. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of the biodegrading enzyme system. We report the production, purification, and characterization of extracellular PHB depolymerase from Stenotrophomonas sp. RZS7 isolated from a dumping yard rich in plastic waste. The isolate produced extracellular PHB depolymerase in the mineral salt medium (MSM) at 30°C during 4 days of incubation under shaking. The enzyme was purified by three methods namely ammonium salt precipitation, column chromatography, and solvent purification. Among these purification methods, the enzyme was best purified by column chromatography on the Octyl-Sepharose CL-4B column giving optimum yield (0.7993 Umg-1mL-1). The molecular weight of purified PHB depolymerase was 40 kDa. Studies on the assessment of biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS7. The results obtained in Fourier-Transform Infrared (FTIR) analysis, High-Performance Liquid Chromatography (HPLC) study and Gas Chromatography Mass-Spectrometry (GC-MS) analysis confirmed the biodegradation of PHB in liquid medium by Stenotrophomonas sp. RZS7. Changes in surface morphology of PHB film in soil burial as observed in Field Emission Scanning Electron Microscopy (FESEM) analysis confirmed the biodegradation of PHB under natural soil environment. The isolate was capable of degrading PHB and it resulted in 87.74% biodegradation. A higher rate of degradation under the natural soil condition is the result of the activity of soil microbes that complemented the biodegradation of PHB by Stenotrophomonas sp. RZS7.


Assuntos
Proteínas de Bactérias/química , Hidrolases de Éster Carboxílico/química , Hidroxibutiratos/química , Plásticos/química , Poliésteres/química , Poluentes do Solo/química , Stenotrophomonas/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Biodegradação Ambiental , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/isolamento & purificação , Cromatografia de Afinidade , Meios de Cultura/química , Humanos , Hidrólise , Peso Molecular , Solo/química , Resíduos Sólidos , Stenotrophomonas/química
6.
Artigo em Inglês | MEDLINE | ID: mdl-31931329

RESUMO

The silkworm, Bombyx mori, is a promising expression system for the production of recombinant proteins, but the purification of these proteins is not easy because of the large amount of host proteins present. To investigate purity, recovery and scale-up ability of the purification of recombinant proteins expressed in silkworm larval hemolymph without any affinity tags, we used mCherry, a red fluorescence protein, as a model. The host cell proteins could be greatly reduced using a three-step chromatography protocol consisting of hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) and heparin chromatography after heat pretreatment. The thermal treatment had the greatest impact on the removal of host cell extracellular proteins and increasing purity. There were still some minor traces of host cell proteins in the purified sample, which showed that the purification of recombinant proteins from the silkworm hemolymph was still challenging. The proposed protocol and affinity tag purification reduced the overall protein content by 99.84% and 99.95%, respectively, while the amount of DNA was reduced by 98.41% and 99.53%, respectively. Purities of our proposed protocol based on SDS-PAGE and capillary electrophoresis (CE) analyses were 85.45% and 43.60%, respectively, while those of Strep-tag affinity purification were 100% or 63.69%, respectively. Using densitometry, the overall recovery was calculated was 5.78%, which was higher than 4.09% using Strep-tag affinity purification. This proposed protocol, mainly based on thermal treatment, HIC, SEC and HiTrap Heparin HP column chromatography, is applicable to an upscalable purification for the silkworm expression system without employing affinity tag chromatography process.


Assuntos
Bombyx/química , Cromatografia de Afinidade/métodos , Hemolinfa/química , Larva/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Bombyx/metabolismo , Eletroforese em Gel de Poliacrilamida , Larva/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
7.
Biomed Chromatogr ; 34(1): e4709, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31630417

RESUMO

Hyaluronidase (Hyal) can be employed to accomplish a diversity of complications related to hyaluronic acid (HA). Hyal contains some classes of catalysts that cleave HA. This enzyme is detected in several human tissues as well as in animal venoms, pathogenic organisms and cancers. Destructive cancer cells regularly increase the CD44 receptor existing in a cell membrane. This receptor acts as an exact receptor for HA, and HA is recognized to motivate the migration, spread, attack and metastasis of cancer cells. Nearly all of the methods used to purify Hyal are highly costly and not proper for industrial applications. This survey aims to review different methods of Hyal purification, which acts as an anticancer agent by degrading HA in tissues and thus inhibiting the CD44-HA interaction. Hyal can be successfully employed in the management of cancer, which is associated with HA-CD44. This review has described different methods for Hyal purification to prepare an origin to develop a novel purification technique for this highly appreciated protein. Using multiple columns is not applicable for the purification of Hyal and thus cannot be used at the industrial level. It is better to use affinity chromatography of anti-Hyal for Hyal with one-step purification.


Assuntos
Cromatografia de Afinidade , Hialuronoglucosaminidase , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Receptores de Hialuronatos/química , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/química , Hialuronoglucosaminidase/isolamento & purificação , Hialuronoglucosaminidase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
8.
Biomed Chromatogr ; 34(1): e4693, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31465544

RESUMO

High-density lipoproteins (HDLs) have anti-inflammatory and antioxidant properties and are potentially cardio-protective. Defective HDL function is caused by alterations in both the proteome and lipidome of HDL particles. As potential biomarkers, the development of analytical methods is necessary for the enrichment of HDLs. Therefore, a method for selective enrichment of HDLs using immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) is presented. SPE-based isolation of HDLs from whole serum is adopted as an alternative to traditional ultracentrifugation methods followed by SDS-PAGE. The enrichment mechanism relies on isoelectric points of lipoproteins and metal oxide. Negatively charged lipoprotein particles interact with positively charged metal oxides and IMAC affinity, which acts as a cation. Identified proteins from HDL through MALDI-MS analysis are apo AI, AII, AIV, CI, CIII, E, J, M, H, serum amyloid A and other nonapoproteins that are part of HDL particles and perform cellular functions. This serum-based proteomics approach gives insight into the functional role of HDL. HDL-associated phospholipids have also been analyzed by LDI-MS. Results suggest that the adopted analytical strategy is a feasible idea to extract lipoproteins from serum. A comparative study of healthy and diseased samples using this approach will provide valuable information in future.


Assuntos
Cromatografia de Afinidade/métodos , Lipoproteínas HDL/sangue , Fosfolipídeos/sangue , Proteoma/análise , Humanos , Lipoproteínas HDL/isolamento & purificação , Fosfolipídeos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
9.
Adv Biochem Eng Biotechnol ; 171: 1-22, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31848634

RESUMO

The use of recombinant DNA methods to produce large quantities of protein for therapeutic uses has revolutionized medicine. Industrial challenges for manufacture of biotherapeutic proteins are related to the characteristics of these proteins and the increasing quantities required to address needs of patients, worldwide. A brief overview of therapies in which proteins are employed helps to frame some of the challenges facing their industrial production. The number of molecules and their applications have significantly expanded over the last 15-20 years, together with the quantities used to address specific indications. Challenges addressed include achieving cell density, protein expression, separations of cells and protein, protein purification, and segmentation of protein production into smaller quantities with the evolution of personalized medicine and products designed for increasingly small patient populations. This chapter highlights some of the current challenges.


Assuntos
Proteínas Recombinantes/uso terapêutico , Cromatografia de Afinidade , Humanos
10.
J Pharm Biomed Anal ; 177: 112895, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31580988

RESUMO

A flower-like gold nanoparticles (FGN)-based immunochromatographic test strip (ICS) was developed and used for the first time for the rapid simultaneous detection of fumonisin B1 (FB1) and deoxynivalenol (DON) in Chinese traditional medicine. Several experimental conditions affecting the sensitivity of ICS have been investigated, including the type of FGN, the preparation conditions of FGN-monoclonal antibody (MAb) conjugates, and the process parameters of ICS. Under the optimal experimental conditions, the visual limit of detection was 5.0 ng/mL (corresponding to 50 µg/kg in Chinese traditional medicine samples) for both FB1 and DON, and detection can be completed within 5 min. In addition, the natural samples were analyzed using high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA), and the results of these methods showed good correlation with those obtained using ICS. The procedure using FGN-based simultaneous ICS was sensitive, rapid, and convenient for on-site detection of a large number of samples.


Assuntos
Carcinógenos Ambientais/análise , Cromatografia de Afinidade/instrumentação , Contaminação de Medicamentos/prevenção & controle , Medicamentos de Ervas Chinesas/análise , Nanopartículas Metálicas/química , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/normas , Fumonisinas/análise , Ouro/química , Limite de Detecção , Tricotecenos/análise
11.
Artigo em Inglês | MEDLINE | ID: mdl-31881515

RESUMO

Polyclonal immunoglobulin therapeutics comprising dosed IgG and IgM combinations are powerful tools in fighting cancer and severe infections. The inability of protein ligands to produce polyclonal IgG- and IgM-enriched formulations and recover monoclonal IgM calls for novel ligands with superior biorecognition activity. In this study, a peptoid ligand discovered by our group, and integrated into affinity adsorbents LigaTrap Technologies' "Human IgG" and "Human IgM", were utilized to purify IgG and IgM from complex fluids. IgG purification from human serum using LigaTrap IgG afforded 94.6% purity and 62.9% yield, on par with Protein A/G resins. When challenged with CHO and HEK cell culture harvests with low IgG titer (<1 mg/mL), LigaTrap IgG returned values of yield and purity well above 60% and 90%. LigaTrap IgM was evaluated for purifying IgM in comparison with commercial adsorbents, and afforded a product purity of 93% from a CHO harvest (IgM titer of 1 mg/mL) and 75.1% yield from a HEK harvest (0.5 mg/mL). LigaTrap-M provided IgM enrichment up to 11-fold higher than HiTrap resin. The peptoid adsorbents separated IgG-depleted human serum into IgM- and IgA-enriched fractions. These results demonstrate the potential of the peptoid ligand for manufacturing polyclonal Ig formulations and monoclonal IgM therapeutics.


Assuntos
Cromatografia de Afinidade/métodos , Imunoglobulina G/isolamento & purificação , Imunoglobulina M/isolamento & purificação , Peptoides , Proteínas Recombinantes/isolamento & purificação , Animais , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Peptoides/química , Peptoides/metabolismo , Proteínas Recombinantes/metabolismo
12.
Anal Bioanal Chem ; 412(5): 1151-1158, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31867701

RESUMO

Tetanus still possesses a high infection risk and leads a number of human deaths in poor nations. Point-of-care and ultrasensitive detection of tetanus antibody levels in serum is the key to decrease the risk of tetanus infection and improve the health of people. In this work, by using ultra bright fluorescent nanospheres (FNs) and portable lateral flow test strip (LFTS), a point-of-care and ultrasensitive sensing method has been developed for the detection of tetanus antibodies in human serum. This assay works quite well for tetanus antibodies in the concentration range from 0.0002 to 0.0220 IU/mL with a low detection limit of 0.00011 IU/mL, which is 100-fold lower than conventional gold-based LFTSs. The high sensitivity makes this method suitable for use to detect the low-abundant target in real samples. Besides, this cost-effective FN-based LFTS assay possesses good selectivity, high accuracy, and satisfactory reliability, which holds great potential as a robust candidate for routine medical diagnosis and rapid home testing. Graphical abstract.


Assuntos
Anticorpos Antibacterianos/sangue , Cromatografia de Afinidade/métodos , Clostridium tetani/imunologia , Corantes Fluorescentes/química , Nanosferas , Sistemas Automatizados de Assistência Junto ao Leito , Tétano/diagnóstico , Humanos , Limite de Detecção , Espectrometria de Fluorescência
13.
Artigo em Inglês | MEDLINE | ID: mdl-31841976

RESUMO

Probing the affinity of a ligand for homologous protein targets currently relies on laborious assays that need special equipment and high amounts of isolated, highly pure proteins. Herein we present the use of pISep, an integrated buffer system and modeling package, as an analytical method to rapidly and accurately probe the binding strength and mechanisms of homologous proteins to surface-bound ligands. To demonstrate our method, we utilized the four subclasses of human immunoglobulin G (IgG) as model homologous protein targets and the IgG-binding peptide HWRGWV as model ligand. Following IgG adsorption on a HWRGWV-Toyopearl adsorbent, the pISep buffer system was used to run uncoupled dual elution gradients of pH (from pH 8.5 to 2.5) and either isocratic or time dependent salt concentration. Both the sequence and partial overlap of elution times (IgG4 > IgG3 ≥ IgG1 > IgG2) was found to match closely the values of binding strength (KD) determined with both in silico docking simulations and isothermal titration calorimetry experiments. pISep gradients performed at different values of ionic strengths provided a means to compare the contribution of hydrophobic vs. electrostatic interactions to the IgG-peptide affinity. The shifts in retention times indicated that, among the various components of the binding energy, the hydrophobic interaction dominates in the binding of IgG2 and IgG4, whereas the binding of IgG1 and IgG3 features a balance of electrostatic and hydrophobic modes. These findings were also confirmed by the in silico analysis of the complexes formed by HWRGWV and the Fc fragment of the IgG subclasses. Collectively, these results indicate that the retention times on pISep elution gradients - in particular peak max, overlap, and shift under different conditions - directly correlate to the strength and nature of protein-ligand interactions. This work demonstrates the effectiveness of the pISep toolbox for probing the differential binding of homologous proteins to a reference ligand and informing the optimization of platform processes for the purification and fractionation of biotherapeutics.


Assuntos
Cromatografia de Afinidade/métodos , Peptídeos , Ligação Proteica , Proteínas Recombinantes , Adsorção , Calorimetria , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Ligantes , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Cloreto de Sódio/química , Eletricidade Estática
14.
Artigo em Inglês | MEDLINE | ID: mdl-31841979

RESUMO

An on-line purification and entrapment system was developed that could extract a protein from a sample such as serum and entrap this protein within a small column for use in high-performance affinity chromatography. Human serum albumin (HSA) was employed as a model protein for this work. Immunoextraction columns containing polyclonal anti-HSA antibodies were developed to capture and isolate HSA from applied samples. This was followed by the use of a strong cation-exchange column to recapture and focus HSA as it eluted from the immunoextraction columns. The recaptured HSA was entrapped within 1.0 cm × 2.1 mm I.D. columns containing hydrazide-activated silica and in the presence of oxidized glycogen as a capping agent. The binding and elution properties of HSA on the various components of this system were examined and optimized. The entrapped columns produced by this system were then evaluated for their use in binding studies with several sulfonylurea drugs. The HSA columns created by this approach typically contained 0.3-0.6 nmol HSA and were stable over several weeks and more than 50-60 sample injections. Drug binding constants could be determined with these columns in 8 min or less by zonal elution and gave good agreement with literature values. The same system could be used for the capture and entrapment of other proteins by utilizing antibodies against the given target for immunoextraction.


Assuntos
Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Preparações Farmacêuticas , Proteínas , Humanos , Preparações Farmacêuticas/análise , Preparações Farmacêuticas/metabolismo , Ligação Proteica , Proteínas/isolamento & purificação , Proteínas/metabolismo , Albumina Sérica Humana
15.
Anal Bioanal Chem ; 411(30): 8153-8162, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31797014

RESUMO

This work describes an analytical procedure based on automated affinity purification followed by liquid chromatography-electrospray tandem mass spectrometry with a conventional triple quadrupole analyzer, in order to detect synthetic insulins (Apidra®, Humalog®, Levemir®, NovoRapid®, and Tresiba®) in human urine. Sample preparation included ultrafiltration followed by immunoaffinity purification on monolithic microcolumns. Chromatographic separation was performed by a C18 microbore column, while mass spectrometric identification of the analytes was achieved by a triple quadrupole mass spectrometer under positive ion electrospray ionization and acquisition mode in selected reaction monitoring. Identification of the synthetic insulins was performed by selecting at least two characteristic ion transitions for each analyte. The newly developed method was validated in terms of specificity, recovery, matrix effect, sensitivity, robustness, and repeatability of retention times and relative ion transition abundance. The specificity and the reproducibility of the relative retention times and the relative abundance of the characteristic ion transitions selected was confirmed to be fit for purposes of ensuring the unambiguous identification of all target analytes, also in the forensic field. The extraction yield was estimated at greater than 60% and the matrix effect smaller than 35%. The lower limits of detection were in the range of 0.02-0.05 ng/mL, proving the method to be sufficiently sensitive to detect the abuse of insulins in cases where they are used as performance-enhancing agents in sport. The applicability of the developed method was assessed by the analysis of urine samples obtained from diabetic subjects treated with Tresiba® and/or Humalog®, whose presence was confirmed in urine samples collected after the administration of therapeutic doses. Graphical abstract A hybrid assay comprising MSIA-based immunoextraction combined with liquid chromatography-electrospray tandem mass spectrometry was developed and validated for the detection of recombinant insulins in human urine.


Assuntos
Cromatografia de Afinidade/métodos , Cromatografia Líquida/métodos , Insulina/urina , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia de Afinidade/instrumentação , Humanos , Limite de Detecção , Proteínas Recombinantes/urina , Reprodutibilidade dos Testes
16.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1134-1135: 121853, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31785532

RESUMO

A box-shaped or cuboid packed-bed device was used for monoclonal antibody (mAb) separation using protein A affinity chromatography. The separation efficiency of the device was compared with an equivalent column i.e. packed with same resin, and having identical bed height and bed volume. The protein A media packed cuboid device had a larger number of theoretical plates than its equivalent column, e.g. 8750/m as opposed to about 4700/m at a flow rate of 0.5 mL/min. In mAb purification experiments, the impurity flow-through and eluted mAb peaks were shaper with the cuboid device. This implied that the effective separation time and buffer consumption with this device was lower, the purified mAb pooled volume was smaller, and the mAb concentration in the pooled volume was greater. Equivalent separation efficiency could be obtained with the cuboid device using higher flow rates than that used with the column. For instance, elution peaks equivalent to those obtainable by the column could be obtained at a 5 times greater flow rate using the cuboid device. The results discussed in this paper clearly demonstrate the potential for improving the efficiency of protein A affinity chromatography based mAb purification by using a cuboid packed-bed device.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Proteína Estafilocócica A/química , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Técnicas de Cultura de Células , Cromatografia de Afinidade/instrumentação , Cricetinae , Cricetulus , Desenho de Equipamento
17.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1130-1131: 121806, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31670106

RESUMO

Lectins are carbohydrate binding proteins with many physiological and biotechnological applications. Isolation of proteins is normally time-consuming and encompasses multiple and, sometimes, complicated steps that hinder reproducibility and yield. Affinity chromatography is an efficient way to simplify and improve protein purification, however often requiring an expensive and fragile stationary phase. In this regard, automated flow-based systems minimize the time for extraction of species from solid samples without hindering the features of batch procedures. In this work, a new inexpensive affinity-based stationary phase was developed for in-line separation of jacalin, a galactose-binding lectin from jackfruit seeds. In the flow manifold, in-line extraction of proteins was also carried out with continuous monitoring using the spectrophotometric Biuret assay. For protein determination, linear response was observed from 3.0 to 15 g L-1. The results of the analysis of protein extracts from jackfruit seeds obtained with the herein described procedure and batch procedure agreed with 95% confidence level. Quantitative extraction of proteins from jackfruit seed powder required recirculation of extraction buffer for 15 min through a lab-made polymethylmethacrylate (PMMA) column containing 200 mg of the crude seed powder. In the chromatographic step, jacalin was isolated after 300 s. Therefore, three essential steps for jacalin isolation were performed in one manifold in a fast way, minimizing sample consumption and solution handling. Additionally, the versatile and multi-task developed flow manifold can be useful for routine analysis and preparative procedures, being adaptable for the extraction and separation of other species from solid matrixes with continuous monitoring of the processes.


Assuntos
Fracionamento Químico/métodos , Cromatografia de Afinidade/métodos , Lectinas de Plantas , Proteínas de Plantas/isolamento & purificação , Artocarpus/química , Lectinas de Plantas/análise , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Proteínas de Plantas/análise , Proteínas de Plantas/química , Projetos de Pesquisa , Sementes/química
18.
Artigo em Inglês | MEDLINE | ID: mdl-31704445

RESUMO

We have recently introduced a non-chromatographic alternative for antibody (Ab) purification, one which does not require the use of Protein A. With this approach, polyclonal human or mouse immunoglobulins (IgG's) are captured almost quantitatively by Tween-20 micelles conjugated with a [chelator:divalent metal cation] complex. Target IgG structure remains native even following extraction from the surfactant aggregate. In the present work, we explore the effect of varying both components of the [metal:chelator] pair on the yield of purified Ab. Capture efficiency is observed to correlate with the formation of sufficiently large detergent aggregates, as determined by dynamic light scattering (DLS) and polyacrylamide gel electrophoresis (PAGE). This, in turn, depends on the rigidity and aromaticity of the chelator. Detergent aggregates are stable over a wide range of pH values (pH = 3-9). Under acidic conditions (3-3.8) they lead to good IgG recovery yields (70-78%) with purity similar to that obtained with Protein A chromatography while maintaining the monomeric state of the IgG's. Finally, the influence of the environment and the presence of various water-soluble chelators (e.g. EDTA, histidine, imidazole) on process efficiency, is described.


Assuntos
Quelantes/química , Imunoglobulina G/isolamento & purificação , Metais/química , Animais , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Micelas , Polissorbatos , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-31704622

RESUMO

Puerariae Radix (PR) serves as food and medicinal plant for thousands of years with explicit efficacy for heart diseases, while biological target specifically binding-oriented screening of the active components in PR remains a preliminary stage. Cell membrane chromatography (CMC) is newly developed approach where interactions between active components and certain biological targets can be effectively studied, Human umbilical vein endothelial cell (HUVEC) membrane, with its abundant receptors such as ß and AT1, is most eligible for constructing CMC. In this study, an HUVEC/CMC-LC-MS2 system was developed for screening active components in PR, 11 compounds were screened out and four of them were identified. Besides puerarin, the rest identified are daidzin, pueroside D and 3'-hydroxypuerarin. The study provides more reference for CMC applications and PR exploitation.


Assuntos
Membrana Celular/metabolismo , Cromatografia de Afinidade/métodos , Medicamentos de Ervas Chinesas/análise , Isoflavonas/análise , Espectrometria de Massas/métodos , Linhagem Celular , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Isoflavonas/química , Isoflavonas/isolamento & purificação , Isoflavonas/metabolismo , Reprodutibilidade dos Testes
20.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1134-1135: 121782, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31778946

RESUMO

Traditional Chinese medicine (TCM) represents a valuable resource for lead compounds discovery. Given the complexity of TCM components, analytical methods play a key role in novel drug development. In our study, we established a high specific and reliable bio-active components screen system, where ß2 adrenergic receptor (ß2-AR) was immobilized on silica by non-covalent bonds and packed into a stainless steel column (4.6 × 50 mm, 7 µm) to form ß2-AR chromatography column. The column was further coupled with high performance liquid chromatography-time of flight tandem mass spectrometry (TOF-MS/MS). By utilizing this strategy, we successfully identified four ß2-AR-targeting compounds: tetrahydroberberine, tetrahydrocolumbamine, fumarine and corydaline from Corydalis Rhizome. The association constants between ß2-AR and tetrahydroberberine (9.04 × 104/M) as well as fumarine (4.30 × 104/M) were determined by frontal chromatography. We also found that these two compounds shared the identical binding site on immobilized ß2-AR with corresponding concentrations of 6.67 × 10-4 M and 5.88 × 10-4 M, respectively. The newly established method represents an efficient tool to identify the target specific natural compounds.


Assuntos
Alcaloides de Berberina , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas , Receptores Adrenérgicos beta 2/metabolismo , Alcaloides de Berberina/análise , Alcaloides de Berberina/metabolismo , Corydalis/química , Avaliação Pré-Clínica de Medicamentos/métodos , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/metabolismo , Receptores Adrenérgicos beta 2/química
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