Direct purification of recombinant hepatitis B core antigen from two different pre-conditioned unclarified Escherichia coli feedstocks via expanded bed adsorption chromatography.

Direct recovery of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli homogenates via expanded bed adsorption chromatography (EBA) has been explored in this study. Streamline DEAE was selected as the anion exchanger to recover HBcAg from heat-treated and non-heat-treated unclarified feedstocks. The use of anion-exchanger for direct extraction of proteins from unclarified feedstock is not preferred due to lack of specificity of its ligand. In this study, thermal treatment of the unclarified feedstock at 60 degrees C has resulted in 1.2- and 1.8-fold increases in yield and purity of HBcAg, respectively, compared with that purified from non-heat-treated feedstock. Heating the crude feedstock has resulted in denaturation and precipitation of contaminants in the feedstock, hence reducing non-specific interactions between the cell debris and adsorbent. The selectivity of the anion-exchanger has also been increased as shown in the breakthrough curve obtained. Enzyme-linked immunosorbent assay showed that the antigenicity of the HBcAg from heat-treated unclarified feedstock is still preserved.

Application of short monolithic columns for improved detection of viruses.

Monolithic chromatography media represent a novel generation of stationary phases introduced in the last 10-15 years providing a chromatography matrix with enhanced mass transfer and hydrodynamic properties. These features allow for an efficient and fast separation of especially large biomolecules like e.g., DNA and viruses. In this study, the enrichment of virus RNA on short monolithic columns prior to molecular detection of viruses is described. Measles and mumps viruses were chosen as model viruses. The results show that it is possible to bind viral RNA on monoliths and concentrate viral nucleic acids from a fairly dilute sample. Consequently, a potential application of short monolithic columns is the concentration of virus RNA to improve the sensitivity and selectivity of viral detection with the possibility of isolating viral RNA from cell-free biological fluids.

Xanthine oxidase-catalyzed metabolism of 2-nitrofluorene, a carcinogenic air pollutant, in rat skin.

The reductive metabolism of 2-nitrofluorene, a carcinogenic air pollutant, in rat skin microsomes and cytosol was investigated. 2-Nitrofluorene was reduced to the corresponding amine by the microsomes with NADPH and by the cytosol with 2-hydroxypyrimidine or 4-hydroxypyrimidine under anaerobic conditions. The cytosolic activity was much higher than that of skin microsomes. The 2- or 4-hydroxypyrimidine-linked nitroreductase activity was inhibited by oxypurinol and (+/-)-8-(3-methoxy-4-phenylsulfinylphenyl) pyrazolo[1,5-a]-1,3,5-triazine-4(1H)-one (BOF-4272), inhibitors of xanthine oxidase, but not by menadione, chlorpromazine and isovanillin, inhibitors of aldehyde oxidase. When skin cytosol was applied to a DEAE-cellulose column, the fractions containing xanthine oxidase exhibited a marked 2-hydroxypyrimidine-linked nitroreductase activity. In contrast, the aldehyde oxidase fraction showed little activity. Nitroreductase fractions obtained by ion exchange chromatography showed a band in Western blotting analysis using anti-rat xanthine oxidase. Moreover, the xanthine oxidase fraction exhibited a significant nitroreductase activity in the presence of 2-hydroxypyrimidine, 4-hydroxypyrimidine or hypoxanthine, and these activities were inhibited by inhibitors of xanthine oxidase. These results indicated that reduction of 2-nitrofluorene in the skin was mainly catalyzed by xanthine oxidase.

Polyallylamine-grafted cellulose gel as high-capacity anion-exchanger.

A new cellulose-based anion-exchanger was prepared by grafting polyallylamine onto cellulose. The material was obtained by partial oxidation of a size-exclusion grade cellulose gel by aq. NaIO4, forming dialdehyde cellulose, followed by Schiff base formation with a polyallylamine (PAA, molecular mass 5000) and subsequent reduction for stabilization. Three grades of PAA-cellulose gels, with amino group contents of 0.78, 1.01 and 1.28 mmol/g cellulose, were examined for their ionic interaction with mono- and divalent carboxylic acids at pH 2.5-5.5. While the retention factor for monovalent acids was nearly proportional to the amino group content of the gel, that for divalent acids was remarkably greater for the PAA-cellulose gel than for the conventional diethylaminoethyl (DEAE) cellulose gel bearing more amino groups (1.97 mmol/g cellulose). Such high capacity can be explained by the high local density of amino groups on grafted PAA, in contrast to the random and sparse charge distribution in conventional exchangers.

[Anti-tuberculous IgE antibodies. I. Immunodominant antigens]. / Protivotuberkuleznye IgE-antitela. I chast'. Immunodominantnye antigeny.

It is widely accepted that protection against tuberculosis is provided by the formation of type 1 immune response, which is characterized by the production of IFN-gamma and IL-2. However, type 2 antimycobacterial immune response is also present: specific IgE antibodies that are IL-4 dependent, are usually found in tuberculosis patients. There is elevated production of type 2 cytokines in some cases. Thus, both types of an immune response can simultaneously develop, probably counteracting with each other. It is unknown which of mycobacterial antigens are capable of inducing a preferential type 2 response. To detect these antigens, the authors studied tuberculosis IgE antibodies in the sera of 500 tuberculosis patients by using the ELISA assay with ultrasonic disintegrated M. Tuberculosis H37Rv (sonicate). Antigens recognized by IgE antibodies were found to be localized in the cell wall of mycobacteria. The IgE-response was specific since the sera did not react with the antigens of atypical mycobacteria and other bacterial species.

Effect of adsorbent porosity on performance of expanded bed chromatography of proteins.

Expanded bed or fluidized bed adsorption has emerged as an important unit operation in downstream processing of proteins. A number of specifically designed commercial adsorbents are available today for expanded bed purification of proteins. Protein purification essentially requires adsorbent matrices that have large pore size. Very large pore size or macroporous adsorbents can provide high efficiency in packed beds even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the macropores. This is reflected in leveling off of HETP (height equivalent to theoretical plate) versus flow curve after a threshold velocity. Expanded bed operation, on the other hand, can also show plateauing of the HETP curve, but not necessarily on account of macroporosity of adsorbent. It is shown in this article how any adsorbent intended for protein adsorption in expanded bed mode can give plateauing HETP curve, regardless of pore size. As a result, RTD measurements on an expanded bed can give equal, and at times better, performance than a corresponding packed bed. Large pore size, on the other hand, can result in lesser retention of biomass and easy flushing of the adsorbent to obtain an entirely particulate-free adsorbent prior to the product elution step. Adsorbent with larger pores is also shown to provide faster and more efficient elution both in packed and expanded bed modes.

Modeling column regeneration effects on ion-exchange chromatography.

The effect of in-place regeneration on equilibrium and kinetic characteristics of the adsorption of bovine serum albumin to a DEAE-cellulose anion exchanger has been determined. Regeneration with sodium hydroxide and time of exposure showed no effect on equilibrium behavior. Breakthrough curves were measured for protein adsorption on fixed-bed columns and analyzed by a simple model to determine the relevant rate constants for the adsorption process. It was found that forward adsorption rate constant decreased exponentially with the chemical treatment exposure time. The implications of the results on the design and optimization of ion-exchange chromatographic processes are discussed.

Aislamiento de isoenzimas de fosfatasa alcalina en plasma humano, por cromatografía de intercambio iónico / Alkaline phosphatase isolation isoenzymes in human plasma ionic chromatography

A partir de la preparación de fosfatasa alcalina (EC 3.1.3.1) parcialmente purificada con etanol, de plasma de personas con grupo 0, se separaron cuatro fracciones con actividad enzimática, mediante cromatografía en columna de DEAE-celulosa, con gradiente discontinuo de NaCI. También se extrajo fosfatasa alcalina de hígado, hueso y mucosa intestinal humanos, a fin de caracterizar las distintas formas moleculares. el uso de inhibidores (L-fenil-alcalina y urea) y electroforesis en gel de poliacrilamida, permitió identificar las isoenzimas presentes en los extractos de tejidos y en los picos de elución cromatográfica de la enzima de plasma. La isoenzima hepática se encuentra en todas las fracciones. La primera eluye con NaCI 40 mM y puede separarse en dos (IA e IB). Ambas contienen las isoenzimas intestinal y ósea, además de formas parcialmente desializadas de la hepática. La segunda fracción (NaCI 60 mM) contiene exclusivamente fosfatasa alcalina hepática. La tercera (NaCI 200 mM), de alto peso molecular, está compuesta por las isoenzimas ósea, intestinal y hepática. El perfil es muy reproducible. La comparación del cromatograma correspondiente a plasma normal con el de un paciente portador de un tumor con metástasis óseas, muestra notables diferencias, indicando la utilidad diagnóstica del método, ya que permite identificar el órgano de origen, en casos de incremento de fosfatasa alcalina en plasma.

Aislamiento de isoenzimas de fosfatasa alcalina en plasma humano, por cromatografía de intercambio iónico / Alkaline phosphatase isolation isoenzymes in human plasma ionic chromatography

A partir de la preparación de fosfatasa alcalina (EC 3.1.3.1) parcialmente purificada con etanol, de plasma de personas con grupo 0, se separaron cuatro fracciones con actividad enzimática, mediante cromatografía en columna de DEAE-celulosa, con gradiente discontinuo de NaCI. También se extrajo fosfatasa alcalina de hígado, hueso y mucosa intestinal humanos, a fin de caracterizar las distintas formas moleculares. el uso de inhibidores (L-fenil-alcalina y urea) y electroforesis en gel de poliacrilamida, permitió identificar las isoenzimas presentes en los extractos de tejidos y en los picos de elución cromatográfica de la enzima de plasma. La isoenzima hepática se encuentra en todas las fracciones. La primera eluye con NaCI 40 mM y puede separarse en dos (IA e IB). Ambas contienen las isoenzimas intestinal y ósea, además de formas parcialmente desializadas de la hepática. La segunda fracción (NaCI 60 mM) contiene exclusivamente fosfatasa alcalina hepática. La tercera (NaCI 200 mM), de alto peso molecular, está compuesta por las isoenzimas ósea, intestinal y hepática. El perfil es muy reproducible. La comparación del cromatograma correspondiente a plasma normal con el de un paciente portador de un tumor con metástasis óseas, muestra notables diferencias, indicando la utilidad diagnóstica del método, ya que permite identificar el órgano de origen, en casos de incremento de fosfatasa alcalina en plasma. (AU)

Evaluation of a simplified microchromatographic technique for hemoglobin A2 determination.

The simplified Hb A2 determination based on microchromatography in Pasteur pipets filled with DEAE-cellulose with glycine-KCN-NaCl as developers [14] is compared with a reference Hb A2 determination procedure based on starch-block electrophoresis. The utility of microchromatography as a routine Hb A2 assay and as a screening method to detect beta-thalassemia trait carriers and patients with iron deficiency anemia was investigated. Day-to-day variation of a control hemolysate and the correlation between the values obtained with the two methods and between determinations in duplicate on the same sample are given. The mean values obtained with both methods for the different groups do not differ significantly but the standard deviations and the coefficients of variation observed by the microchromatography are generally higher. Microchromatography in Pasteur pipets tends to overestimate low and normal Hb A2 concentrations and to underestimate high Hb A2 concentrations. The results of microchromatography are more significant for the diagnosis when Hb A2 concentrations are expressed in weight hemoglobin per volume of blood and not in percentages. The microchromatographic procedure was recently marketed. The results obtained with the commercial columns were in good correlation with those obtained with starch-block electrophoresis, but commercial columns give a 18% overestimation of the Hb A2 concentrations.

Cascade chromatography and automated multidimensional fractionation.

A general method of automated multidimensional fractionation has been developed. Its basic ingredients are: (a) cascade fractionation, i.e., the sequential fractionation of components obtained from each chromatographic dimension on the same or a different dimension, (b) on-line acquisition and processing of data at each stage in the fractionation procedure, (c) a method for determining the beginning and end of each peak during column elution, and (d) automatic linkage of the successive stages in a chemical fractionation scheme based on information obtained before or during each stage. Apparatus for automatic cascade chromatography and conditional fractionation is described. The method can be extended to provide completely automatic separation of pure components from complex mixtures.