The solid phase attachment of fungal hyphae in an ELISA to screen for antifungal antibodies

A method to immobilize fungal hyphae onto the wells of 96-well microplates for use in an in-direct ELISA to screen for antifungal antibodies in sera and cell culture supernatants is described. The hyphae from three genera (Penicillium, Eurotium andFusarium) were successfully attached by overnight drying onto wells precoated with poly-L-lysine and glutaraldehyde. Microscopy revealed that the hyphae remained attached to the wells throughout the ELISA and antiserum titrations showed that the attached hyphae were uniformly coated and remained reactive. Background absorbances were low and the plates could be stored at –20 °C without loss of reactivity.     

An avidin-biotin solid phase ELISA for femtomole isopentenyladenine and isopentenyladenosine measurements in HPLC purified plant extracts

A solid phase enzyme immunoassay was developed for isopentenyladenine (iP) and isopentenyladenosine (iPA) quantitation in HPLC purified plant extracts. It was performed on antigen-coated microtitration plates on which bound antibodies were indirectly labeled by the means of a biotinylated goat anti-rabbit antibody and an avidin-alkaline phosphatase conjugate. Less than 3 femtomoles of iP or iPA were easily detected and the measuring range extended from 3 femtomole to 1 picomole. The reproducibility has been tested and intra- and interassay variations did not exceed 5.0%. The specificity of iPA antibodies was good, as determined by cross-reactivity measurements with other adenylic compounds. The specificity of the measurements for iP and iPA was demonstrated by analysis of the immunoreactivity of fractions obtained by HPLC separation of a methanolic tobacco leaf extract.     

Cellulose acetate electrophoresis of casein proteins.

Samples of the milk proteins α_s1-casein and β-casein partially dephosphorylated by means of bovine spleen phosphoprotein phosphatase have been electrophoretically analysed using cellulose acetate as the supporting medium and Procion blue as the protein dye. Sufficient resolution was obtained in 1 hr to allow quantification of the proteins present. Skimmed-milk samples and acid-precipitated whole casein samples have been analysed by the same technique. The advantages of the method are discussed in relation to the more conventional electrophoretic techniques normally used to analyse these milk proteins.     

Cellulose acetate impregnated with polyacrylamide: a novel medium for electrophoresis

Cross-linked polyacrylamide gel containing a low proportion of methylenebis-acrylamide has been incorporated into cellulose acetate membranes. Unlike on cellulose acetate itself, electrophoresis on these modified membranes enables molecular sieving of proteins under a wide range of conditions. By modifying only part of the membrane, samples can be loaded in the normal way and sharpen as they migrate across the cellulose acetate-polyacrylamide boundary. The thin membranes retain their general ease of handling and speed of staining and destaining.     

Cellulose acetate membrane electrophoresis and FTIR spectroscopy as methods of identifying a fucoidan in Fucusvesiculosus Linnaeus

A fucoidan from brown algae such as the common bladder wrack (Fucusvesiculosus Linnaeus) is now widely examined in many countries for its interesting biological and therapeutic properties. In this study, the fucoidan was identified during extraction in hydrochloric acid; its presence was confirmed by FTIR spectroscopy. Two slightly different structures were found in real samples of dried bladder wrack supplied by Flos and Witherba, by comparing them with a reference sample of F. vesiculosus L. A simple, repeatable analytical procedure was developed using apparatus for cellulose acetate membrane electrophoresis and this was supplemented by semi-quantitative analysis.     

Flea diversity as an element for persistence of plague bacteria in an East African plague focus

Plague is a flea-borne rodent-associated zoonotic disease that is caused by Yersinia pestis and characterized by long quiescent periods punctuated by rapidly spreading epidemics and epizootics. How plague bacteria persist during inter-epizootic periods is poorly understood, yet is important for predicting when and where epizootics are likely to occur and for designing interventions aimed at local elimination of the pathogen. Existing hypotheses of how Y. pestis is maintained within plague foci typically center on host abundance or diversity, but little attention has been paid to the importance of flea diversity in enzootic maintenance. Our study compares host and flea abundance and diversity along an elevation gradient that spans from low elevation sites outside of a plague focus in the West Nile region of Uganda (~725-1160 m) to higher elevation sites within the focus (~1380-1630 m). Based on a year of sampling, we showed that host abundance and diversity, as well as total flea abundance on hosts was similar between sites inside compared with outside the plague focus. By contrast, flea diversity was significantly higher inside the focus than outside. Our study highlights the importance of considering flea diversity in models of Y. pestis persistence.     

Cellulose acetate entrapment of Escherichia coli on cotton cloth for aspartate production

Summary Aspartase containingEscherichia coli cells were entrapped in cellulose acetate aggregated on cotton cloth. The enzyme activity of the cloth was stabilized by treatment with polyethylenimine and alkaline glutaraldehyde in the presence of 0.1 M sodium dithionite. A column packed with the cloth segments catalyzed 100% conversion of 1 M ammonium fumarate to aspartic acid at a space velocity of 7/h. When the same cloth segments were stirred at 100 rpm in the substrate solution, the productivity of the cloth increased 2.5 times.     

Enzyme-linked immunosorbent assay for Met-enkephalin using unconjugated enkephalin as a solid phase antigen

This paper presents a competitive enzyme-linked immunosorbent assay for Met-enkephalin, in which there is no need for preparing any special derivative of Met-enkephalin for its optimal function as a solid-phase antigen. Immunoplate wells were first coated with free Met-enkephalin, and then incubated with antiserum and free Met-enkephalin. The antibodies bound to the solid-phase Met-enkephalin were determined by using anti-IgG conjugated to horseradish peroxidase. The detection limit of the assay was 44 fmol Met-enkephalin per well. The results also showed that Met-enkephalin bound to the solid phase can be titrated directly.     

Ethyl acetate production in the gas phase

Summary Ethyl acetate was produced from gaseous ethanol and acetic acid using a lipase from Porcine Pancreas at 25 °C. Its production was investigated at different moisture content of lipase enzyme powder because it is known that moisture content of enzyme affects the reaction in the gas phase.     

Amyloid-beta as a "difficult sequence" in solid phase peptide synthesis

The phenomenon of "difficult sequence" has long frustrated chemists in their efforts to assemble peptides that contain such sequences by solid phase synthesis methods. A variety of remedial measures are available to minimize or even abolish the negative impact of these sequences during synthesis. These include the use of elevated temperatures and stronger acylating reagents. Amyloid-beta, a fragment of the amyloid precursor protein, contains 40-43 residues and possesses a C-terminal sequence that is particularly resistant to ready solid phase synthesis making it a "difficult sequence" peptide. This review focuses on approaches to successfully assemble the peptide by both Boc- and Fmoc solid phase synthesis.     

Evaluation of COMU as a coupling reagent for in situ neutralization Boc solid phase peptide synthesis

Benzotriazole‐based coupling reagents have dominated the last two decades of solid phase peptide synthesis. However, a growing interest in synthesizing complex peptides has stimulated the search for more efficient and low‐cost coupling reagents, such as COMU which has been introduced as a nonexplosive alternative to the classic benzotriazole coupling reagents. Here, we present a comparative study of the coupling efficiency of COMU with the benzotriazole‐based HBTU and HCTU for use in in situ neutralization Boc‐SPPS. Difficult sequences, such as ACP(65–74), Jung–Redeman 10‐mer, and HIV‐1 PR(81–99), were used as model target peptides on polystyrene‐based resins, as well as polyethylene glycol‐based resins. Coupling yields obtained using fast in situ Boc‐SPPS cycles were determined with the quantitative ninhydrin test as well as via LC‐MS analysis of the crude cleavage products. Our results demonstrate that COMU coupling efficiency was less effective compared to HBTU and HCTU with HCTU ≥ HBTU > COMU, when polystyrene‐based resins were employed. However, when the PEG resin was employed in combination with a safety catch amide (SCAL) linker, more comparable yields were observed for the three coupling reagents with the same ranking HCTU ≥ HBTU > COMU. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support.

Approaches to direct solid phase sequencing of genomic and plasmid DNA have been developed using magnetic beads, coated with streptavidin, as solid support. The DNA is immobilized through selective incorporation of biotin into one of the strands. A single stranded template, suitable for sequencing, is obtained through strand-specific elution. Using this concept, in vitro amplified plasmid DNA and chromosomal DNA were sequenced directly from single colonies. The solid phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The system was found to be suitable for sequencing using both isotope- and fluorescent-labelled primers.     

Macroporous hydrophobic cloth (polymacron) as a solid phase for nucleic acid probe hybridizations

Polyester cloth (polymacron) was employed as a solid phase onto which polymerase chain reaction (PCR) products from theListeria monocytogenes hlyA gene were dot blotted and sensitively detected by hybridization with nucleic acid probes. This inexpensive solid phase exhibited a high DNA binding capacity and provided ease of handling and washing between each reaction step, resulting in simple hybridization procedures for PCR product detection.Contribution No. 95-004     

Bacterial Cellulose as a Matrix for Microorganisms in Bioelectrocatalytic Systems

Applied Biochemistry and Microbiology - Bacterial cellulose (BC) produced by the Komagateibacter sucrofermentas VKPM B-11267 bacteria was used as a carrier for immobilization of acetic acid...     

High molecular weight neoglycoconjugates for solid phase assays

Adsorption of a carbohydrate on solid phase is the necessary stage of the immunosorbent assay (ELISA) and analogous methods of the study of carbohydrate-protein interaction. Usually physical adsorption on polystyrene requires a high concentration of conjugated carbohydrate and, thus, enormous consumption of it. In this study, we explored two approaches allowing more rational use of oligosaccharide (Glyc). The first of them is based on the covalent immobilization of neoglycoconjugates on the NH₂-modified polystyrene; the second one is based on the elevated adherence of high m.w. neoglycoconjugates to polystyrene. Covalent immobilization of polyacrylamide conjugates, Glyc-PAA, provided a possibility to solve the problem, but the nonspecific binding of antibodies in ELISA proved to be unacceptably high. At the same time, the increase of the Glyc-PAA m.w. from 30 kDa to 2,000 kDa allowed a 10–20 fold decrease of its consumption, when using physical adsorption, whereas the assay background remained at the low level. The amount of 2,000 kDa Glyc-PAA that is sufficient for the coating of a standard 96-well plate corresponds to the nanomole level of oligosaccharide, this providing a possibility to use saccharides that are available in a very limited amount when studying the carbohydrate-protein interaction with solid-phase techniques. Published in 2005.     

Rapid fluorometric detection for completeness in solid phase coupling reactions

The fluorescamine test for the rapid detection of trace amounts of uncoupled products from solid phase peptide synthesis is reported. This novel procedure can detect much smaller amounts of incomplete coupling with greater simplicity than has previously been possible. Since the test is carried out under mild conditions certain side reactions are circumvented. The fluorophor-resins are easily viewed under long wave ultraviolet light, are stable at room temperature, and may be used for quantitative evaluation.