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At least 343 records · Page 19Linked to original sources
PubMed2001

Glass fibers covered with sol-gel glass as a new support for affinity chromatography columns: a review.

The search for mechanical supports for biochemically active compounds serving as immunochemical sensors has been the goal of many studies. A new compound in the form of gel fiberglass (GFG) membranes was recently developed as an example of such supports. In this review, these membranes were analyzed with respect to their use for cancer detection. The membranes are prepared from glass fibers covered with oxysilane to provide a sol-gel glass matrix. Derivatization of the support eliminates nonspecific adsorption. A thin layer of protein trapped in the gel glass during its preparation is deposited on the surface of a lattice of glass fibers. The major innovation of the membrane is its large area. External agents percolating through such a membrane contact a maximal number of molecules of the compounds trapped in the sol-gel glass. As a result, this membrane is highly effective. Each GFG column is built from a series of 20 to 30 membranes. The preparation of such columns is relatively simple, requiring only several hours. The capacity of GFG columns is high: the total amount of tumor-associated antigens (TAA) isolated by these columns from the blood of colon cancer patients reached 50% of the total protein and amounted to up to 9-12 mg/ml of serum. The main components of the isolated TAA were the soluble p66 and p51 proteins. A determination of their concentration by HPLC can be used for early cancer detection. Thus, the described method allows the easy and highly effective isolation of TAA and can be used for different goals, including cancer diagnosis. GFG supports have great potential for the isolation of various macromolecules utilizing specific ligands.

Antigens↗
PubMed1985

Purification of oligomeric staphylococcal alpha-toxin by affinity chromatography on digitonin-sepharose.

An effective concentration of alpha-toxin from Staphylococcus aureus Wood 46, directly from the culture supernatant, could be achieved by adsorption on digitonin-sepharose and elution with 3 mol/l sodium thiocyanate (NaSCN). The toxin was further purified by gelchromatography. The purified product yielded 1 single protein band upon SDS-polyacrylamide electrophoresis. It was nonhemolytic, but reacted with anti-alpha-toxin under complement fixation. Dialysis against 0.14 mol/l NaCl with hydrophobic amino acids partially reactivated the alpha-hemolytic activity of the toxin. Ultracentrifugal analysis yielded sedimentation coefficients for the purified toxin of approximately 3,7 S when dissolved in 3 mol/l NaSCN and of about 12 S after dialysis against 0.14 mol/l NaCl (Table 1). The spontaneous oligomerization of the alpha-toxin during dialysis against 0.14 mol/l NaCl possibly resulted from a change in configuration induced by its adsorption to digitonin-sepharose.

Bacterial Toxins↗
PubMed2001

Affinity chromatography of branched oligosaccharides in rat liver beta-glucuronidase.

Rat liver microsomal and lysosomal beta-glucuronidase-derived glycopeptides were obtained by extensive Pronase digestion followed by N-[14C]acetylation and desialylation by neuraminidase treatment. These glycopeptides were studied by sequential chromatography on lectin-affinity columns such as concanavalin A, lentil lectin, Phaseolus vulgaris erythroagglutinin, Ricinus communis agglutinin I, Triticum vulgaris agglutinin, Glycine max agglutinin and Ulex europaeus agglutinin. Using serial lectin affinity chromatography approach combined with neuraminidase treatment allowed us to show the unexpected presence of complex tri- and/or tetraantennary type glycans (40.8 and 17.0% for microsomal and lysosomal enzyme, respectively). Moreover, the application of neuraminidase treatment revealed that complex biantennary type glycans, present on lysosomal beta-glucuronidase, are almost fully sialylated while the same type of glycans present on microsomal enzyme do not contain sialic acid. Furthermore, the results obtained confirmed that microsomal and lysosomal beta-glucuronidases possess high mannose and/or hybrid type glycans (19.6 and 36.6%, respectively), and complex biantennary type glycans (38.9 and 46.4%, respectively).

Animals↗
PubMed2003

Purification of native dehydrin from Glycine Max cv., Pisum sativum, and Rosmarinum officinalis by affinity chromatography.

An improved method for the purification of dehydrin from soy (glycine max) is described. Acidic extraction of soy whey was followed by a three step chromatographic process: capture on copper charged Chelating Sepharose Big Beads, intermediate hydrophobic interaction chromatography on Source 15 PHE, and a polishing step on blue Sepharose. The 32-kDa native soy dehydrin was purified to a purity of greater than 98.5% with an overall recovery of 63%. When compared to a previously published purification procedure, recovery, time requirements, and sample preparation steps were improved. The developed method is readily scaleable. Preliminary results show that the process can be used for dehydrins from rosemary (Rosmarinum officinalis) and pea (Pisum sativum).

Blotting, Western↗
PubMed1989

[Purification of human thrombin by affinity chromatography for its use in preparations for biological coagulation].

Biological glue is obtained by mixing different specific plasma proteins including a serine protease, thrombin. Surprisingly at present the thrombin used in such a mixture is from equine or bovine origin while all other components are from human. In this paper we described a particular efficient and specific chromatographic method for the purification of human thrombin usable as a serine protease in the preparation of biological glue. A pure and active thrombin is obtained from a plasma fraction after adsorption on benzamidine-Spherodex followed by an elution with non specific (sodium chloride gradient) or biospecific competitors (arginine methylester or benzamidine). The obtained thrombin with a yield close to 80% and a purification factor close to 160, showed good properties in the replacement of animal thrombin in the condition of biological glue.

Benzamidines↗
PubMed2003

Expanded bed adsorption processing of mammalian cell culture fluid: comparison with packed bed affinity chromatography.

A comparison between expanded bed adsorption and conventional packed bed Protein A Fast Flow to purify the anti-rHBsAg mAbs from feedstock is presented in this work. Direct capture by STREAMLINE expanded bed adsorption chromatography resulted in 92% product recovery and sevenfold more concentrated product with similar purity levels compared to that obtained by the standard packed method. The process time and buffer consumption were reduced in the expanded bed adsorption method not only with the binding-elution conditions but also with the use of NaOH during the cleaning-in-place step. The latter is the most widely accepted agent in downstream processing, being a cost effective technique that provides not only efficient cleaning but also sanitizes complete column systems and destroys pirogens.

Adsorption↗
PubMed1996

Affinity chromatography with immunochemical detection applied to the analysis of human methionyl granulocyte colony stimulating factor in serum.

An on-line, automated, HPLC method was developed for the separation and detection of recombinant human methionyl granulocyte colony stimulating factor (GCSF) and GCSF modified with poly(ethylene glycol) (PEG-GCSF) in rat serum. The automated method consists of an initial immunoaffinity chromatography step, a microbore reverse phase separation, and postcolumn immunochemical detection. Calibration curves were constructed with a lower limit of 1.5 ng of GCSF (80 fmol) and 7.5 ng of PEG-GCSF in 120 microL of rat serum. In vivo serum samples from rats dosed with PEG-GCSF were also analyzed. The development of the method is discussed as well as its general application to the detection of metabolites of recombinant proteins in body fluids.

Animals↗