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Selected AbstractsComparison of ATP and in vivo bioluminescence for assessing the efficiency of immunomagnetic sorbents for live Escherichia coli O157:H7 cellsJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2002W. Sun Aims:,To develop methods to assess the efficiency of immunomagnetic separation (IMS). Methods and Results:,The capturing efficiency of biosorbents for Escherichia coli O157:H7, constructed using streptavidin-coated magnetic beads and biotinylated antibodies, was tested using both in vivo and ATP bioluminescence. Both methods were suitable for the enumeration of bacteria captured by the biosorbents. The level of both ATP and in vivo bioluminescence depended on the media used, but was unaffected by the magnetic beads. The capture efficiency depended on time and sample volume, but did not depend on the length of spacer arm of the biotinylation agent. For cell concentrations of , 105 cfu ml,1, in a 1-ml sample volume, nearly 80,85% recovery of the pathogen was observed after 0·5 h of incubation. For an 11-ml sample containing 104 cfu ml,1, maximum recovery (50% of cells) was achieved only after 2 h incubation. Conclusions:,The detection limit of an ATP-based bioluminescent assay for E. coli O157:H7 was reduced by 1 log cycle after optimization of IMS. The bioluminescent methods could be used for screening and testing the affinity of antibodies or other affinity elements of biosorbents towards live bacterial cells. Significance and Impact of the Study:,Bioluminescent assays provide an easy way to optimize conditions for the capture of bacteria by biosorbents in real time. [source] Protein adsorption and platelet adhesion of polysulfone membrane immobilized with chitosan and heparin conjugatePOLYMERS FOR ADVANCED TECHNOLOGIES, Issue 2 2003M.-C. Yang Abstract Polysulfone (PSF) membranes were treated with ozone to introduce peroxides, and then grafted with either acrylic acid or chitosan, followed by the immobilization of heparin. The effect of spacer arm on blood compatibility was investigated using three chitosans of different molecular weight [1170 (water soluble), 160,000, and 400,000] and similar degrees of deacetylation (75%). The hydrophilicity was evaluated by measuring the contact angle of water. Blood compatibility was evaluated using the activated partial thromboplastin time (APTT) as well as the adhesion of platelets. The protein affinity was determined by the absorption of human serum albumin (HSA) and human plasma fibrinogen (HPF). The results show that by the coupling of chitosan, the amount of heparin immobilized can be increased by four times. Water contact angle (from 78,° to 41,°) decreased with the increase of the amount of heparin immobilized, showing increased wettability. The heparinized PSF membrane showed longer APTT and decreasing platelet adhesion, compared to that of unmodified PSF membrane. The adsorption of HSA and HPF were reduced to 17 and 6%, respectively. This suggests that longer spacer binding to heparin can increase the opportunity of anti-coagulation on contacting blood. These results demonstrated that the hydrophilicity and blood compatibility of PSF membrane could be improved by chitosan and heparin conjugate. Copyright © 2003 John Wiley & Sons, Ltd. [source] High-performance affinity chromatography with immobilization of protein A and L-histidine on molded monolithBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2002Quanzhou Luo Abstract Reactive monoliths of macroporous poly(glycidyl methacrylate- co -ethylene dimethacrylate) have been prepared by "in-situ" copolymerization of the monomers in the presence of porogenic diluents. Protein A and L-histidine were immobilized on the monoliths directly or through a spacer arm, respectively. The properties of these two kinds of affinity columns were characterized, and the results showed that the columns with coupling of ligands by a spacer arm have some extent of non-specific adsorption for bovine serum albumin. The affinity column based on the monolithic polymer support provided us with good hydrodynamic characteristic, low flow resistance, and easy preparation. These two affinity columns were used for the purification of immunoglobulin G from human serum. The purity of the purified IgG was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The stability of the protein A affinity column was investigated, and its performance remained invariable after half a year. The effects of the nature and the pH of the buffer system on the adsorption capacity of human IgG on histidyl affinity column were also investigated. The protein A affinity column is favorable for rapid analysis of human IgG samples. In contrast, the advantages of mild elution conditions, high stability, as well as low cost provide the histidyl column further potential possibility for fast removal of IgG from human plasma in clinical applications. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 481,489, 2002. [source] Influence of peptide ligand surface density and ethylene oxide spacer arm on the capture of porcine parvovirusBIOTECHNOLOGY PROGRESS, Issue 5 2009Caryn L. Heldt Abstract In previous work, we identified two trimeric peptide ligands (designated WRW and KYY), which bound specifically to porcine parvovirus (PPV) and demonstrated their ability to capture and remove the virus from solutions containing 7.5% human blood plasma. This article examines the influences of peptide density and the presence of an ethylene oxide spacer arm on the efficiency of virus capture using these two ligands. The WRW peptide bound the most virus from plasma solutions at the lowest peptide density tested (0.008 mmol/g dry resin), and binding was enhanced by the presence of the spacer arm. On the other hand, the KYY peptide bound the most viruses at the same low peptide density, but it performed better in the absence of the spacer arm. Of the two, the binding efficiency of the WRW peptide was more sensitive to peptide density and spacer arm presence. These results indicate that low peptide densities enhance binding selectivity, facilitating specific peptide-virus binding even in the presence of plasma proteins which can theoretically bind nonspecifically. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | ||||||||||