RESUMEN
In this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by high-intensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO2) selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX), and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a high-resolution mass spectrometer (LTQ-Orbitrap XL). The strategy was optimized for the global phosphoproteome analysis of Jurkat T-cells. Using this accelerated workflow, HIFU-TiO2-SCX-LC-MS/MS, 15,367 phosphorylation sites from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins can be efficiently identified in less than 15 h.
Asunto(s)
Fosfopéptidos/análisis , Fosfoproteínas/análisis , Proteómica/métodos , Fraccionamiento Químico/métodos , Cromatografía por Intercambio Iónico/economía , Cromatografía por Intercambio Iónico/métodos , Humanos , Células Jurkat , Fosfopéptidos/aislamiento & purificación , Fosfoproteínas/aislamiento & purificación , Fosforilación , Proteoma/análisis , Proteoma/aislamiento & purificación , Proteómica/economía , Espectrometría de Masas en Tándem/economía , Espectrometría de Masas en Tándem/métodos , Factores de Tiempo , Titanio/química , Flujo de TrabajoRESUMEN
In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex™ AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac™ PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35-1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis. Graphical abstract Xylanase activity assay automation.
Asunto(s)
Aspergillus niger/enzimología , Cromatografía por Intercambio Iónico/métodos , Endo-1,4-beta Xilanasas/metabolismo , Pruebas de Enzimas/métodos , Geobacillus stearothermophilus/enzimología , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/economía , Endo-1,4-beta Xilanasas/análisis , Pruebas de Enzimas/economía , Hidrólisis , Límite de Detección , Factores de Tiempo , Xilanos/metabolismoRESUMEN
B-phycoerythrin (B-PE) was separated and purified from microalga Porphyridium cruentum using one-step chromatographic method. Phycobiliproteins in P. cruentum was extracted by osmotic shock and initially purified by ultrafiltration. Further purification was carried out with a SOURCE 15Q exchange column and analytical grade B-PE was obtained with a purity ratio (A545/A280) of 5.1 and a yield of 68.5%. It showed a double absorption peaks at 545 nm and 565 nm and a shoulder peak at 498 nm, and displayed a fluorescence emission maximum at 580 nm. The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a bulky band between 18 and 20 kDa which could be assigned to subunits α and ß and a low intensity band of 27 kDa assigned to γ subunit. Our protocol provides attractive alternative to consider for the purification procedure to obtain analytical grade B-PE at commercial level.
Asunto(s)
Cromatografía por Intercambio Iónico , Ficoeritrina/aislamiento & purificación , Porphyridium/química , Resinas de Intercambio Aniónico/química , Cromatografía por Intercambio Iónico/economía , Cromatografía por Intercambio Iónico/métodos , Electroforesis en Gel de Poliacrilamida , Ficoeritrina/química , UltracentrifugaciónRESUMEN
An ion chromatography-electrospray ionization-tandem mass spectrometry (IC-ESI-MS/MS) method has been developed for rapid and accurate measurement of perchlorate in polar snow and ice core samples in which perchlorate concentrations are expected to be as low as 0.1 ng L(-1). Separation of perchlorate from major inorganic species in snow is achieved with an ion chromatography system interfaced to an AB SCIEX triple quadrupole mass spectrometer operating in multiple reaction monitoring mode. Under optimized conditions, the limit of detection and lower limit of quantification without pre-concentration have been determined to be 0.1 and 0.3 ng L(-1), respectively, with a linear dynamic range of 0.3-10.0 ng L(-1) in routine measurement. These represent improvements over previously reported methods using similar analytical techniques. The improved method allows fast, accurate, and reproducible perchlorate quantification down to the sub-ng L(-1) level and will facilitate perchlorate measurement in the study of natural perchlorate production with polar ice cores in which perchlorate concentrations are anticipated to vary in the low and sub-ng L(-1) range. Initial measurements of perchlorate in ice core samples from central Greenland show that typical perchlorate concentrations in snow dated prior to the Industrial Revolution are about 0.8 ng L(-1), while perchlorate concentrations are significantly higher in recent (post-1980) snow, suggesting that anthropogenic sources are a significant contributor to perchlorate in the current environment.
Asunto(s)
Monitoreo del Ambiente/métodos , Hielo/análisis , Percloratos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Contaminantes Químicos del Agua/análisis , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/economía , Cromatografía por Intercambio Iónico/métodos , Monitoreo del Ambiente/economía , Límite de Detección , Espectrometría de Masa por Ionización de Electrospray/economía , Espectrometría de Masas en Tándem/economía , Espectrometría de Masas en Tándem/métodosRESUMEN
A fast, accurate and precise ion chromatography method with pulsed amperometric detection was applied to evaluate a variety of parameters affecting the determination of total iodine in serum and urine of 81 subjects, including 56 obese and 25 healthy Polish children. The sample pretreatment methods were carried out in a closed system and with the assistance of microwaves. Both alkaline and acidic digestion procedures were developed and optimized to find the simplest combination of reagents and the appropriate parameters for digestion that would allow for the fastest, least time consuming and most cost-effective way of analysis. A good correlation between the certified and the measured concentrations was achieved. The best recoveries (96.8% for urine and 98.8% for serum samples) were achieved using 1ml of 25% tetramethylammonium hydroxide solution within 6min for 0.1ml of serum/urine samples. Using 0.5ml of 65% nitric acid solution the best recovery (95.3%) was obtained when 7min of effective digestion time was used. Freeze-thaw stability and long-term stability were checked. After 24 weeks 14.7% loss of iodine in urine, and 10.9% in serum samples occurred. For urine samples, better correlation (R(2)=0.9891) of various sample preparation procedures (alkaline digestion and application of OnGuard RP cartidges) was obtained. Significantly lower iodide content was found in samples taken from obese children. Serum iodine content in obese children was markedly variable in comparison with the healthy group, whereas the difference was less evident when urine samples were analyzed. The mean content in serum was 59.12±8.86µg/L, and in urine 98.26±25.93 for obese children when samples were prepared by the use of optimized alkaline digestion reinforced by microwaves. In healthy children the mean content in serum was 82.58±6.01µg/L, and in urine 145.76±31.44µg/L.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Yodo/sangre , Yodo/orina , Obesidad/sangre , Obesidad/orina , Adolescente , Niño , Cromatografía por Intercambio Iónico/economía , Femenino , Humanos , Masculino , Sensibilidad y EspecificidadRESUMEN
Advances in molecular biology and cell culture technology have led to monoclonal antibody titers in excess of 10 g/L. Such an increase can pose concern to traditional antibody purification processes due to limitations in column hardware and binding capacity of Protein A resins. Recent development of high capacity cation exchangers can make cation exchange chromatography (CEX) a promising and economic alternative to Protein A capture. This work investigates the feasibility of using CEX for direct capture of monoclonal antibodies from high titer cell culture fluids. Two resin candidates were selected from seven newer generation cation exchangers for their higher binding capacity and selectivity. Two monoclonal antibodies with widely differing pI values were used to evaluate the capability of CEX as a platform capture step. Screening of loading pH and conductivity showed both resins to be capable of directly capturing both antibodies from undiluted cell culture fluid. At appropriate acidic pH range, product loading of over 65 g/L resin was achieved for both antibodies. A systematic design of experiment (DOE) approach was used to optimize the elution conditions for the CEX step. Elution pH showed the most significant impact on clearance of host cell proteins (HCPs). Under optimal conditions, HCP reduction factors in the range of 9-44 were achieved on the CEX step based on the pI of the antibody. Apart from comparing CEX directly to Protein A as the capture method, material from either modality was also processed through the subsequent polishing steps to compare product quality at the drug substance level. Process performance and product quality was found to be acceptable using the non-affinity based process scheme. The results shown here present a cheaper and higher capacity generic capture method for high-titer antibody processes.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Cromatografía por Intercambio Iónico/métodos , Biotecnología/métodos , Técnicas de Cultivo de Célula , Cromatografía por Intercambio Iónico/economía , Costos y Análisis de Costo , Tecnología Farmacéutica/métodosRESUMEN
Human plasma is dominated by high-abundance proteins which severely impede the detection of low-abundance proteins. Unfortunately, now there is no efficient method for large-scale depletion of high-abundance proteins in human plasma. In this study, we developed a new strategy, strong anion exchange (SAX)/RP 2D LC system, which has potential for large-scale depletion of high-abundance proteins in human plasma. Separation gradients of the system were optimized to ensure an extensive separation of plasma proteins. Plasma was fractionated into 67 fractions by SAX. All these fractions were subjected a thorough separation by the 2D RPLC and 66 peaks with high UV absorption (>20 mAU) at 215 nm were collected. Proteins in these peaks were identified by LC-MS/MS analysis. Results showed that 83 proteins could be identified in these peaks, 68 among them were reported to be high- or middle-abundance proteins in plasma. All these proteins had definite retention times and were mapped in the 2D SAX-RP system, which resulted in accurate depletion of high-abundance proteins with ease. Our studies provide a convenient and effective method for large-scale depletion of high-abundance proteins and in-depth research in human plasma proteomics.
Asunto(s)
Proteínas Sanguíneas/aislamiento & purificación , Cromatografía por Intercambio Iónico/métodos , Proteómica/métodos , Aniones/química , Cromatografía por Intercambio Iónico/economía , Humanos , Proteómica/economía , Espectrometría de Masas en Tándem/economía , Espectrometría de Masas en Tándem/métodos , Factores de TiempoRESUMEN
A simple, rapid, precise and specific isocratic HPAE-PAD method for quantification of CGP69669A was developed and validated. CGP69669A is a glycomimetic of sialyl Lewis(x) and an antagonist of E-selectin with potential application in the treatment of inflammatory skin disease. Quantification was performed using a Dionex CarboPac(TM) PA-200 anion-exchange column (3 × 250 mm) with 100 mm NaOH solution as mobile phase, a flow rate of 0.50 mL/min and an injection volume of 10 µL. A quadruple potential waveform was used to detect the carbohydrate (+0.1 V from 0.00 to 0.40 s, -2.0 V from 0.41 to 0.42 s, +0.6 V at 0.43 s and -0.1 V from 0.44 to 0.50 s with current integrated between 0.20 and 0.40 s for detection) and rafinose was employed as an internal standard. The optimized conditions enabled rapid elution of CGP69669A (at 3.0 min) without interference from solvent peaks or substances present in the skin. The method showed good intra- and inter-day precision and accuracy and the response was linear from 1.0 to 25 µg/mL. This is the first validated direct method for the quantification of CGP69669A. It will now be employed in studies investigating the topical and transdermal delivery of CGP69669A in vitro and in vivo and it should also be of use for other applications of this molecule.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Oligosacáridos/farmacocinética , Piel/metabolismo , Animales , Cromatografía Líquida de Alta Presión/economía , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/economía , Selectina E/metabolismo , Límite de Detección , Modelos Lineales , Oligosacáridos/análisis , Absorción Cutánea , PorcinosRESUMEN
A simple method for the speciation analysis of bio-accessible arsenic (As) in rice was developed using a continuous on-line leaching method to release the bio-accessible fraction. The continuous on-line leaching method has several advantages over commonly used batch methods including quicker and easier sample preparation, reduced risk of contamination and access to real time leaching data. The bio-accessibility of As in the samples was monitored using inductively coupled plasma mass spectrometry (ICP-MS). Results from a certified reference material as well as cooked and uncooked white rice showed that the majority of As was leached by saliva. Results obtained using the continuous on-line leaching method were comparable to those obtained using a batch method. Speciation analysis of the saliva leachate was performed using ion exchange chromatography coupled to ICP-MS. The four most toxic forms of As (As(III), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and As(V)) were clearly separated within 5 min in a single chromatographic run. Over 92% of bio-accessible As in the certified reference material and uncooked white rice sample was in the form of DMA and As(V), whereas it was present as DMA and As(III) in the cooked white rice.
Asunto(s)
Arsénico/análisis , Cromatografía por Intercambio Iónico/instrumentación , Espectrometría de Masas/instrumentación , Oryza/química , Arsénico/aislamiento & purificación , Cromatografía por Intercambio Iónico/economía , Diseño de Equipo , Espectrometría de Masas/economía , Reproducibilidad de los ResultadosRESUMEN
The objective of this study was to develop a simple method of phosvitin purification from hen egg yolk without using organic solvents. Egg yolk was diluted with equal volume of water and stirred for one hour at room temperature, followed by centrifugation to remove soluble proteins along with most of the yolk lipids in the supernatant. The granules were collected as the precipitate containing minimum amount of lipids (dry granules). The dry granules were dissolved in 0.05 M carbonate-bicarbonate buffer at pH 9.6, which yields a light yellowish solution used for anion exchange chromatography. Phosvitin fraction was collected from anion exchange chromatography as the last eluting peak with a purity of 92.6% and a yield of 35.4% of total phosvitin in the yolk or a recovery of 1.9% of total yolk dry matter, which are comparable to current methods employing organic solvents or chromatography after salt fractionation and dialysis. This method developed is simple and fast without using organic solvents.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Yema de Huevo/química , Fosvitina/aislamiento & purificación , Animales , Aniones/química , Pollos , Cromatografía por Intercambio Iónico/economía , Factores de TiempoRESUMEN
Desmosine crosslinks are responsible for the elastic properties of connective tissues in lungs and cardiovascular system and are often compromised in disease states. We developed a new, fast, and simple cation exchange HPLC assay for the analysis of desmosine and isodesmosine in animal elastin. The method was validated by determining linearity, accuracy, precision, and desmosines stability and was applied to measure levels of desmosines in porcine and murine organs. The detection and quantification limits were 2 and 4 pmol, respectively. The run-time was 8 min. Our cation exchange column does not separate desmosine and isodesmosine, but their level can be quantified from absorbance at different wavelengths. Using this assay, we found that desmosines levels were significantly lower in elastin isolated from various organs of immunodeficient severe combined immunodeficiency mice compared with wild-type animals. We also found that desmosines levels were lower in lung elastin isolated from hyperhomocysteinemic Pcft(-/-) mice deficient in intestinal folate transport compared with wild-type Pcft(+/+) animals.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/métodos , Desmosina/análisis , Elastina/química , Isodesmosina/análisis , Animales , Cromatografía Líquida de Alta Presión/economía , Cromatografía por Intercambio Iónico/economía , Hiperhomocisteinemia/metabolismo , Límite de Detección , Pulmón/química , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Inmunodeficiencia Combinada Grave/metabolismo , Porcinos , Factores de TiempoRESUMEN
Heparin binding (HB) proteins mediate a wide range of important cellular processes, which makes this class of proteins biopharmaceutically important. Engineering HB proteins may bring many advantages, but it necessitates cost effective and efficient purification methodologies compared to currently available methods. One of the most important classes of HB proteins are fibroblast growth factors (FGFs) and their receptors (FGFRs). In this study, we report an efficient off-column purification of FGF-1 from soluble fractions and purification of the D2 domain of FGFR from insoluble inclusion bodies, using a weak Amberlite cation (IRC) exchanger. FGF-1 and the D2 domain have been expressed in Escherichia coli and purified to homogeneity using IRC resin. This approach is an alternative to conventional affinity column chromatography, which exhibits several disadvantages, including time-consuming experimental procedures for purification and regeneration and results in the expensive production of recombinant proteins. Results of the heparin binding chromatography and steady state fluorescence experiments show that the FGF-1 and the D2 are in a native conformation. The findings of this study will not only aid an in-depth investigation of this class of proteins but will also provide avenues for inexpensive and efficient purification of other important biological macromolecules.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Factor 1 de Crecimiento de Fibroblastos/aislamiento & purificación , Heparina/química , Receptores de Factores de Crecimiento de Fibroblastos/aislamiento & purificación , Cromatografía por Intercambio Iónico/economía , Factor 1 de Crecimiento de Fibroblastos/genética , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
We investigated and optimized a purification process, suitable for industrial scale, to obtain pharmaceutical grade apo-Tf (apo-transferrin), preserving its physiological properties and functions. Apo-Tf was obtained from fraction IV subfraction 1 and IV subfraction 4 (fraction IV-1,4), a waste product of the Cohn fractionation process, performing a single chromatographic run and two viral inactivation/removal steps. The structural integrity and the biological activity of the final product were extensively tested. The yield of apo-Tf produced was 80% on laboratory scale and 90% in scale-up lots, and the purity was higher than 95%. The purified protein preserves iron- and receptor-binding activities and shows a normal glycosylation pattern. The single chromatographic step process presented here provides an efficient means to prepare commercial quantities of the protein. The final product is sterile and two viral inactivation/removal steps were introduced into the process.
Asunto(s)
Apoproteínas/aislamiento & purificación , Proteínas Sanguíneas/aislamiento & purificación , Fraccionamiento Químico/métodos , Cromatografía por Intercambio Iónico/métodos , Transferrina/aislamiento & purificación , Apoproteínas/metabolismo , Proteínas Sanguíneas/metabolismo , Proliferación Celular , Cromatografía por Intercambio Iónico/economía , Células HeLa , Humanos , Hierro/metabolismo , Unión Proteica , Estabilidad Proteica , Transferrina/metabolismoRESUMEN
Basic operation principles of a lightweight, low power, low cost, portable ion chromatograph utilizing open tubular ion chromatography in capillary columns coated with multi-layer polymeric stationary phases are demonstrated. A minimalistic configuration of a portable IC instrument was developed that does not require any chromatographic eluent delivery system, nor sample injection device as it uses gravity-based eluent flow and hydrodynamic sample injection adopted from capillary electrophoresis. As a detection device, an inexpensive commercially available capacitance sensor is used that has been shown to be a suitable substitute for contactless conductivity detection in capillary separation systems. The built-in temperature sensor allows for baseline drift correction typically encountered in conductivity/capacitance measurements without thermostating device. The whole instrument does not require any power supply for its operation, except the detection and data acquisition part that is provided by a USB port of a Netbook computer. It is extremely lightweight, its total weight including the Netbook computer is less than 2.5kg and it can be continuously operated for more than 8h. Several parameters of the instrument, such as detection cell design, eluent delivery systems and data treatment were optimized as well as the composition of eluent for non-suppressed ion chromatographic analysis of common inorganic cations (Na(+), NH(4)(+), K(+), Cs(+), Ca(2+), Mg(2+), transition metals). Low conductivity eluents based on weakly complexing organic acids such as tartaric, oxalic or pyridine-2,6-dicarboxylic acids were used with contactless capacitance detection for simultaneous separation of mono- and divalent cations. Separation of Na(+) and NH(4)(+) cations was optimized by addition of 18-crown-6 to the eluent. The best separation of 6 metal cations commonly present in various environmental samples was accomplished in less than 30min using a 1.75mM pyridine-2,6-dicarboxylic acid and 3mM 18-crown-6 eluent with excellent repeatability (below 2%) and detection limits in the low micromolar range. The analysis of field samples is demonstrated; the concentrations of common inorganic cations in river water, mineral water and snow samples were determined.
Asunto(s)
Cromatografía por Intercambio Iónico/instrumentación , Cationes/análisis , Cromatografía por Intercambio Iónico/economía , Nieve/química , Agua/análisisRESUMEN
Nitrous acid degradation of heparin followed by high-performance anion-exchange chromatography (HPAEC) separation and ultraviolet matrix assisted laser desorption/ionization time-of-flight (UV-MALDI-TOF) analysis led to the structural determination of six sulfated oligosaccharides. Three different matrices (alpha-cyano-4-hydroxycinnamic acid (CHCA), nor-harmane, and dihydroxybenzoic acid (DHB)) have been used, and the complementary results obtained allowed in most cases to assign the position of sulfate groups. Based on the different cleavages produced on the purified oligosaccharides in source during the MS analysis by the use of the different matrices, this approach provides a new tool for structural analysis.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Heparina/química , Oligosacáridos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión/economía , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/economía , Datos de Secuencia Molecular , Estructura Molecular , Ácido Nitroso/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/economía , Espectrofotometría Ultravioleta , Factores de TiempoRESUMEN
Chromatographic performances are highly influenced by operational parameters. New ion exchangers have tailored matrices providing low backpressure and allowing high flow velocity. By systematic frontal analysis and selectivity determination at different flow rates, we suggested an independent evaluation of major anion exchangers to facilitate media selection, and investigated the relationship between (i) surface modification and (ii) chromatographic performances. Structure-extended resins showed higher binding capacities compared to resins with conventional ligands directly attached to the matrix. Moreover, they maintained mainly high capacities even with extremely high flow velocities. Ligand accessibility was therefore largely enhanced, allowing proteins to interact and bind under harsh conditions. High throughput resins can be used for purification of high volume and high concentration feedstock in limited time. This results in higher productivity and could contribute to cost reduction.
Asunto(s)
Cromatografía por Intercambio Iónico/instrumentación , Cromatografía por Intercambio Iónico/métodos , Resinas Sintéticas/química , Animales , Bovinos , Cromatografía por Intercambio Iónico/economía , Conalbúmina/química , Conalbúmina/aislamiento & purificación , Unión Proteica , Resinas Sintéticas/economía , Albúmina Sérica/química , Albúmina Sérica/aislamiento & purificaciónRESUMEN
BACKGROUND & OBJECTIVE: beta-thalassaemia is a genetic disorder and an important health problem around the world. Quantitative haemoglobin A(2) (HbA(2)) levels are used for the diagnosis of beta-thalassaemia. The conventional methods are high performance liquid chromatography (HPLC), electrophoresis, and microcolumn chromatography techniques. We established a fast protein liquid chromatography (FPLC) method, to measure quantitatively of HbA(2) levels, and compared its efficacy with conventional methods. METHODS: The FPLC method, using a DEAE Sepharose, Hi Trap anion-exchange column chromatography technique was set up for HbA(2) measurement. In this study, 220 blood samples were screened for haemoglobin type by FPLC technique and also using HPLC, microcolumn chromatography and electrophoresis. RESULTS: The FPLC results were highly correlated (r = 0.985, P<0.001) with those of HPLC for quantification of HbA(2) as well as cellulose acetate electrophoresis (r = 0.977) and microcolumn chromatography (r = 0.980). The FPLC method showed 100 per cent sensitivity and specificity, positive and negative predictive value for beta-thalassaemia diagnosis. In addition, the FPLC method was simple, rapid, low cost and reproducible. The HbA(2)/E range of FPLC for beta-thalassaemia was 6-10 per cent, HbE trait was 10-40 per cent, beta-thalassaemia/HbE was 40-60 per cent and homozygous HbE was more than 60 per cent. INTERPRETATION & CONCLUSION: Our findings suggested that FPLC method could be used as a cost-effective method for routine beta-thalassaemia diagnosis.
Asunto(s)
Cromatografía Liquida/métodos , Cromatografía Liquida/normas , Electroforesis/normas , Hemoglobinas/análisis , Hemoglobinas/aislamiento & purificación , Talasemia beta/diagnóstico , Adulto , Cromatografía por Intercambio Iónico/economía , Cromatografía por Intercambio Iónico/métodos , Cromatografía por Intercambio Iónico/normas , Cromatografía Liquida/economía , Análisis Costo-Beneficio , Electroforesis/economía , Electroforesis/métodos , Hemoglobina Fetal/análisis , Hemoglobina Fetal/aislamiento & purificación , Hemoglobina A2/análisis , Hemoglobina A2/aislamiento & purificación , Hemoglobina E/análisis , Hemoglobina E/aislamiento & purificación , Humanos , Tamizaje Masivo/economía , Tamizaje Masivo/métodos , Tamizaje Masivo/normas , Valor Predictivo de las Pruebas , Sensibilidad y EspecificidadRESUMEN
Affinity chromatography on immobilized Protein A is the current method of choice for the purification of monoclonal antibodies (mAbs). Despite its widespread use it presents certain drawbacks, such as ligand instability, leaching, toxicity and high cost. In the present work, we report a new procedure for the purification of two human monoclonal anti-HIV (human immunodeficiency virus) antibodies (mAbs 2G12 and 4E10) from transgenic tobacco plants using stable and low cost chromatographic materials. The first step of the mAb 2G12 purification procedure is comprised of an aqueous two-phase partition system (ATPS) for the removal of polyphenols while providing an essential initial purification boost (2.01-fold purification). In the second step, mAb 2G12 was purified using cation-exchange chromatography (CEX) on S-Sepharose FF, by elution with 20mM sodium phosphate buffer pH 7.5, containing 0.1M NaCl. The eluted mAb was directly loaded onto an immobilized metal affinity chromatography column (IMAC, Zn(2+)-iminodiacetic acid-Sepharose 6B) and eluted by stepwise pH gradient. The proposed method offered 162-fold purification with 97.2% purity and 63% yield. Analysis of the antibody preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme immunosorbent assay (ELISA) and western blot showed that the mAb 2G12 was fully active and free of degraded variants, polyphenols and alkaloids. The effectiveness of the present purification protocol was evaluated by using a second transgenic human monoclonal anti-HIV mAb 4E10. The results showed that the same procedure can be successfully used for the purification of mAb 4E10. In the case of mAb 4E10, the proposed method offered 148-fold purification with 96.2% purity and 36% yield. Therefore, the proposed protocol may be of generic use for the purification of mAbs from transgenic tobacco plants.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Cromatografía de Afinidad/métodos , Anticuerpos Anti-VIH/aislamiento & purificación , Nicotiana/química , Plantas Modificadas Genéticamente/química , Anticuerpos ampliamente neutralizantes , Cromatografía de Afinidad/economía , Cromatografía por Intercambio Iónico/economía , Cromatografía por Intercambio Iónico/métodos , Humanos , Metales/química , Plantas Modificadas Genéticamente/inmunología , Control de Calidad , Nicotiana/genética , Nicotiana/inmunologíaRESUMEN
BACKGROUND: The diverse physiological and pathological role of nitric oxide in innate immune defenses against many intra and extracellular pathogens, have led to the development of various methods for determining nitric oxide (NO) synthesis. NO metabolites, nitrite (NO2-) and nitrate (NO3-) are produced by the action of an inducible Anopheles culicifacies NO synthase (AcNOS) in mosquito mid-guts and may be central to anti-parasitic arsenal of these mosquitoes. METHOD: While exploring a plausible mechanism of refractoriness based on nitric oxide synthase physiology among the sibling species of An. culicifacies, a sensitive, specific and cost effective high performance liquid chromatography (HPLC) method was developed, which is not influenced by the presence of biogenic amines, for the determination of NO2- and NO3- from mosquito mid-guts and haemolymph. RESULTS: This method is based on extraction, efficiency, assay reproducibility and contaminant minimization. It entails de-proteinization by centrifugal ultra filtration through ultracel 3 K filter and analysis by high performance anion exchange liquid chromatography (Sphereclone, 5 mu SAX column) with UV detection at 214 nm. The lower detection limit of the assay procedure is 50 pmoles in all midgut and haemolymph samples. Retention times for NO2- and NO3- in standards and in mid-gut samples were 3.42 and 4.53 min. respectively. Assay linearity for standards ranged between 50 nM and 1 mM. Recoveries of NO2- and NO3- from spiked samples (1-100 muM) and from the extracted standards (1-100 muM) were calculated to be 100%. Intra-assay and inter assay variations and relative standard deviations (RSDs) for NO2- and NO3- in spiked and un-spiked midgut samples were 5.7% or less. Increased levels NO2- and NO3- in midguts and haemolymph of An. culicifacies sibling species B in comparison to species A reflect towards a mechanism of refractoriness based on AcNOS physiology. CONCLUSION: HPLC is a sensitive and accurate technique for identification and quantifying pmole levels of NO metabolites in mosquito midguts and haemolymph samples that can be useful for clinical investigations of NO biochemistry, physiology and pharmacology in various biological samples.
Asunto(s)
Anopheles/química , Nitratos/análisis , Óxido Nítrico/metabolismo , Nitritos/análisis , Animales , Anopheles/clasificación , Anopheles/metabolismo , Cromatografía Líquida de Alta Presión/economía , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/economía , Cromatografía por Intercambio Iónico/métodos , Hemolinfa/química , Óxido Nítrico Sintasa de Tipo II/metabolismo , Sensibilidad y Especificidad , Estómago/químicaRESUMEN
BACKGROUND: Current methods for determination of carbohydrate-deficient transferrin (CDT) are based on separation of the CDT fraction by ion-exchange chromatography on minicolumns and quantification by immunoassay. Alternatively, the transferrin isoforms can be separated by HPLC anion-exchange chromatography and quantified by absorbance. This method has been reported to improve the validity of CDT as a marker of chronic alcohol abuse. METHODS: HPLC on either MonoQ or ResourceQ anion-exchange columns was used to separate and quantify isoforms of transferrin with detection at 460 nm. The result was expressed as the percentage of the disialo form (pI 5.7) of total transferrin (DST). The commercial CDTect assay was used as a comparison method. Serum samples from nondrinkers (n = 57), moderate drinkers (n = 77), and heavy drinkers (n = 139) were analyzed. RESULTS: In ROC analysis for differentiation between moderate and heavy drinkers, the area under the curve (AUC) for the HPLC method was 0.87 (95% confidence interval, 0.81-0.93), whereas that for CDTect was 0.72 (95% confidence interval, 0.64-0.80). At 90% specificity, the sensitivity of DST was 63% (95% confidence interval, 53-73%) compared with 33% (22-44%) for CDT. The reference interval of the HPLC method was 0.68-1.7%. CONCLUSIONS: The HPLC anion-exchange method for quantification of CDT provides substantially better separation between moderate and heavy drinkers than the CDTect method.