Cellulose Discs Research Articles

Studies on the interaction of a surface-bound ligand with a multi-valent high-molecular-weight ligate : The biotinycellulose—avidin system

Two types of biotinylated cellulose disks were examined: filter-paper disks to which biotin had been covalently attached directly to the paper surface (biotinylcellulose) and disks on which biotin was attached to polyacrylamide side-chains grafted onto the filter-paper surface (biotinylpolyacrylamide—cellulose). The amount of avidin taken up from solution by these disks was linearly related to the avidin input concentration and could be estimated by exposure to [ 14C]biotin. The avidin-binding capacity of the disks depended on the surface density of covalently attached ligand and exhibited hyperbolic, Langmuir-type behaviour for both types of disks. The [ 14C]biotin binding capacity of avidinylated disks, on the other hand, showed anomalous, biphasic behaviour: at higher ligand densities, a decrease in [ 14C]biotin binding was observed. The largest anomalies were obtained with biotinylpolyacrylamide—cellulose disks. Calculated ratios of bound [ 14C]biotin vs. amount of avidin tetramer (B/A 4) showed a similar biphasic behaviour. A constant value of B/A 4 = 3 was obtained at low ligand densities, whereas B/A 4 decreased monotonously with increasing ligand density and asymptotically approached B/A 4 = 1. The data could be explained by assuming that at high ligand densities tetrameric avidin interacts with more than one surface-bound biotin residue.

Improved assay for quantitating adherence of ruminal bacteria to cellulose.

A quantitative technique suitable for the determination of adherence of ruminal bacteria to cellulose was developed. This technique employs adherence of cells to cellulose disks and alleviates the problem of nonspecific cell entrapment within cellulose particles. By using this technique, it was demonstrated that the adherence of Ruminococcus flavefaciens FD1 to cellulose was inhibited by formaldehyde, methylcellulose, and carboxymethyl cellulose. Adherence was unaffected by acid hydrolysates of methylcellulose, glucose, and cellobiose.

Coupled reactions for the determination of analytes and enzymes based on the use of luminescence.

Immobilized enzymes are widely used in the clinical laboratory in the assay of several analytes and enzymes. The use of immobilized enzymes makes these reagents recoverable and re-usable, and in most cases increases their stability and catalytic activity. In conjunction with bioluminescent enzymes (firefly and bacterial luciferases) and chemiluminescent catalyst (peroxidase) we set up high-sensitive flow methods based on the use of nylon tube coil or epoxy methacrylate column as solid support. All the NAD(P)/NAD(P)H-dependent dehydrogenases (bacterial luciferase), ATP-dependent enzymes (firefly luciferase) and oxidases producing H2O2 (peroxidase) can be immobilized and a large variety of analytes have been sensitively measured. As an alternative format we also reported a dry chemistry method in which all the enzymes, substrates and cofactors are ready to use, supported on dry cellulose disks. Methodological problems such as flow conditions, stability, pH, ionic strength and analytical performances are also reported.

ChemInform Abstract: Simultaneous Multiple Synthesis of Protected Peptide Fragments on ′Allyl′‐Functionalized Cellulose Disc Supports.

Abstract The synthesis of several protected peptide fragments on cellulose discs1 carrying allylic anchor groups is described. The model peptides include the majority of problematic amino acid residues and those types of side chain protecting groups currently used in the Fmoc-strategy.

Copper Uptake by Ryegrass Seedlings; Contribution of Cell Wall Adsorption

Net copper uptake by cellulose discs, isolated root cell walls, and by live and dead roots of whole ryegrass seedlings, were studied using 64Cu as a tracer. Uptake by cellulose discs stopped after around 10 h while uptake by isolated root cell walls continued for up to 50 h. An initial fast phase of uptake consisting predominantly of cell wall adsorption was similar in live and dead tissue for up to 19 h. A slower phase of uptake continued for up to 50 h, greater in live than in dead tissue, the slower phase of uptake in live tissue consisting of both a living and a dead component. Based on these results, an alternative to the desorption method for estimating the apoplastic contribution to total copper uptake is presented. Time-course studies with seedlings given a variety of growing solution/uptake solution regimes, and the relationship between copper uptake and external copper concentration, for short (4-8 h) and long (42-4 h) term uptakes, suggest that differing contributions of cell wall adsorption and symplasmic absorption may be responsible for differing effects of external copper concentration on uptake being expressed by the same tissue. Water flux had little effect on total uptake of copper although a possible effect on absorption could not be ruled out.

Multidisc RAST: a reliable method for screening of specific IgE antibodies in serum?

"Multidiscs" (md) are cellulose discs to which several allergens are coupled, allowing the simultaneous screening of several specific IgE antibodies. We compared RAST performed with md to RAST performed with the corresponding individual allergens coated on single discs (sd). One hundred and twenty-two RAST positive samples were analyzed for four different allergen groups: moulds, cereals, weed pollens and tree pollens. We found lower RAST sensitivity when screening with mds than with the individual sds in all allergen groups except cereals. In 30 to 70% of the samples, the results were concordant. In 25.5 to 66.7%, md values were lower and in 1.3 to 26.3% higher than sd values. When only qualitative results were considered, the loss of sensitivity appeared acceptable: only 6.7 to 16.7% of the sd positive samples were not detected by md screening. A cost-benefit analysis, calculated on local data, confirmed economic advantages.

Determination of solid-state fungal growth by Fourier transform infrared-photoacoustic spectroscopy

Photoacoustic spectroscopy (PAS) does not require optically transparent samples and is, therefore, well suited for analysis of solid-state samples. Fourier transform infrared (FTIR)-PAS of solid materials containing protein exhibited strong absorption in the amide I and amide II regions of the IR spectrum. Growth of a filamentous fungus, Phanerochaete chrysosporium, on cellulose discs was quantitatively determined by monitoring amide I absorption with FTIR-PAS.

Simultaneous multiple peptide synthesis under continuous flow conditions on cellulose paper discs as segmental solid supports

A new approach to the simultaneous chemical synthesis of large numbers of different peptide sequences is described. 1 The particular features of the method presented 2 are: - cellulose paper discs serve as solid supports for Individual peptide sequences, - the peptide chains are linked to the cellulose via a p-alkoxybenzyl ester anchor cleavable by mild treatment with trifluoroacetic acid in dichloromethane, - the syntheses of peptides are accomplished using N-α-Fmoc protected amino acids, - all reactions are carried out in a simple multi-column continuous flow device with the cellulose discs tightly stacked in column reactors, - as many as 100 discs can be reacted simultaneously with the same amino acid derivative in one reaction column.

Simultaneous multiple synthesis of protected peptide fragments on ‘allyl’-functionalized cellulose disc supports

The synthesis of several protected peptide fragments on cellulose discs 1 carrying allylic anchor groups is described. The model peptides include the majority of problematic amino acid residues and those types of side chain protecting groups currently used in the Fmoc-strategy.

An investigation of moisture sorption in microcrystalline cellulose using sorption isotherms and dielectric response

Measurements were made of the enthalpy changes, using sorption isotherms, and of the changes in dielectric response of microcrystalline cellulose discs when different amounts of water were added. The results are explained by means of a molecular model in which the water is sorbed in a step-wise process of hydrogen bonding.

Studies of human IgE to a sulfonamide determinant

We tested the hypothesis that patients who experience immediate hypersensitivity reactions to sulfonamides (SM) express IgE that can bind to a N 4-sulfonamidoyl determinant (N 4-SM). Sulfamethoxazole (SMX) was coupled to CNBr-activated cellulose disks to form a matrix predominantly substituted with isourea-linked N 4-SMX determinants. After incubation of human sera with these disks or bovine serum albumin substituted disks as a control, the binding of IgE was assessed with 125I-labeled antihuman IgE. The binding ratios (counts per minute SMX disks per counts per minute bovine serum albumin disks) for sera from nonallergic donors and newborn infants averaged 1.11 (± 0.21 SD). Sera from 10 patients with histories of apparent immediate hypersensitivity reactions to SM were studied. Ratios ⩾2.1 (> 4 SD above control) were detected in 70% (seven of 10). Significant binding was detected in the sera of three of seven patients with other forms of SM allergy. Preincubation with SMX (80 mmol/L) inhibited binding 7% to 35% in eight of the 10 positive sera tested. Binding of one highly reactive serum was significantly inhibited by SMX, sulfamethizole, and sulfamerazine, but not sulfanilic acid or trimethoprim. The results of this study suggest that N 4-SM is a major determinant recognized by IgE to SM and that an in vitro assay capable of detecting IgE to SM has been developed.

Irrelevant IgE Binding to Cellulose Discs in RAST

This study describes two sera that showed irrelevant IgE binding to all allergen-coated cellulose discs tested. This irrelevant IgE binding was not observed when allergen extracts were coupled to agarose beads. Such irrelevant IgE binding can be detected using human serum albumin-coated discs.

13] Solid-phase assay for d-biotin and avidin on cellulose disks

This chapter describes the solid-phase assay for d-biotin and avidin on cellulose disks. Most biotin assay procedures exploit the absolute growth requirement of certain bacterial and yeast strains for d-biotin, or depend on the extremely high affinity of avidin for this vitamin. The binding assays, particularly those employing immobilized avidin, are faster and easier to perform. However, when avidin is covalently linked to particulate supports, it becomes difficult to distribute accurately constant amounts of the immobilized protein among different samples and to avoid losing some of the biotin-immobilized-avidin complex while centrifuging or filtering particles to remove unbound biotin. These problems can be overcome by changing the geometry of the avidin-carrying support. The filter paper disks of uniform size, loaded with essentially constant amounts of immobilized avidin, are easily transferable from one aqueous environment to another. Such avidinylated disks of known biotin-binding capacity form the basis of the sequential competition assay in which primary immersion of the disks in a sample of unknown biotin content is followed by exposure to an excess of radioactive biotin. In the event that disks can bind more biotin than test samples contain, decreased disk capacity for the labeled ligand becomes a direct measure of sample biotin content. The source of avidin for preparation of avidin–(biotin–cellulose) disks can be commercial avidin or egg white.

Computer management of oligonucleotide synthesis on cellulose filters.

A computer program, OLIGO, is described which was designed to manage projects involving the synthesis of large numbers of oligonucleotide fragments using cellulose discs as immobilizing support. The program performs the functions of data entry, verification, analysis and modification; the sorting of filters before each step and project status updating after each reaction step; output of the project status at any point and storage of the finalized project data in a permanent reference file.

Preparation and biochemical manipulation of mRNAs and cDNAs on small oligo(dT)-cellulose discs.

Preparation and biochemical manipulation of mRNAs and cDNAs on small oligo(dT)-cellulose discs.

Mesure de la sensibilite des bacteries marines heterotrophes au zinc et au cadmium par la methode de l'antibiogramme. Analyse de la concordance entre les concentrations minimales inhibitrices et les zones d'inhibition sur milieu solide.

Abstract Evaluation of the sensitivity of marine heterotrophic bacteria to heavy metals. Analysis of the concordance between minimal inhibitory concentrations and inhibition zones on solid medium. Sensitivity of bacteria to heavy metals can be estimated by diffusion in solid media or dilution in liquid or solid media. Antibiograms can only be used for metals if well standardized and grounded on the knowledge of the conccordance between inhibitory zones and minimal inhibitory concentrations measured in liquid media. This concordance has been analysed for cadmium and zinc, with several marine bacteria. It was largely depending on whether the inhibitory zones were read. The relationship between CMI and the width of inhibition zones was log‐linear for both metals. A semi‐quantitative scheme is proposed for interpreting antibiograms, using cellulose disks in defined cultural conditions.

A fast and simple enzyme immunoassay method for determination of total IgE in human serum

The measurement of total IgE serum requires sensitive methods such as radioimmunoassays or enzymoimmunoassays. Until now, the immunoenzymatic techniques described used as solid supports: polystyrene tubes [ 11, magnetic polyacrylamideagarose beads [2], cellulose discs [3], polystyrene balls [4], or polystyrene microtitration plates [5]. Here, we describe a fast and sensitive sandwich enzymoimmunoassay, in which the solid-phase consists of polypropylene tubes. The tubes are treated with a solution of glutaraldehyde [6,7] and coated with rabbit immunoglobulins to human IgE. Peroxidase labelled sheep immunoglobulins to human IgE or conjugate and a highly sensitive chromogen, 2,2’-azino-di-(3-ethylbenzothiazolin-6-sulphonate) are used. The manipulation is fast (3.30 h) and the sensitivity of the assay is 0.1 IU of IgE per tube.

Particle counting immunoassay of immunoglobulin E antibodies after their elution from allergosorbents by pepsin: an alternative to the radioallergosorbent test

The absorption of specific antibodies on allergen-coated cellulose discs, followed by pepsin digestion to release the IgE Fc″ fragment, which is then assayed by particle counting immunoassay (PACIA), is a new practical and reliable method to quantitate IgE antibodies and to standardize allergens. The coefficient of variation of repeated assays does not exceed 12.8% and the correlation coefficient with RAST was r = 0.96. Some discrepancies could be explained by differences in the antiserum specificities and the presence of anti-IgE autoantibodies. The determination of IgE antibodies by PACIA offers the following advantages: (I) no radioisotopes; (2) stability of reagents, since antibody-coated latex particles and the standards keep their activity for more than 1 yr; (3) low cost of reagents due to ease of preparation—PACIA does not require purified antibodies to measure IgE but only F(ab′) 2 fragments of the IgG fraction of the anti-IgE antiserum to coat latex; (4) avoidance of nonspecific absorption of the labeled anti-IgE on the solid phase; (5) prevention by elution of the inaccuracies observed in RAST at very high titers of IgE antibodies; (6) expression of the results in international units per milliliter to calculate the ratio of specific IgE Ab vs total IgE, with a high ratio suggesting that the patient is sensitive to a few allergens; (7) standardization of allergenic extracts; and (8) the particular specificity of the anti-IgE antiserum directed against a heat- and protease-resistant fragment of IgE.

A solid-phase immunofluorescence assay (SIFA) using membrane filters

A solid-phase immunofluorescence assay (SIFA) is presented which allows the demonstration of antibodies against soluble antigens. The test is performed by successively incubating nitrate cellulose discs in microtitration plates with anti-Ig, the sample to be tested and FITC-labeled antigen. The fluorescence intensity of the discs is then measured by using a microfluorometer. The assay is simple, needs a small amount of material and allows to test a large number of samples.

Hypersensitivity to chemicals. Correlation of tartrazine hypersensitivity with characteristic serum IgD and IgE immune response patterns.

SummaryA radioimmunoassay inhibition test (CARIAT) has been developed to measure human anti‐tartrazine IgD and IgE antibodies. Graphical analysis of the data obtained has shown that patients with clinically identified tartrazine hypersensitivity can be distinguished from controls by singular characteristics of their IgD but not their IgE antibody patterns. Clinical diagnoses of tartrazine hypersensitivity correlate with relative levels of serum IgD antibody specific for a soluble conjugate of tartrazine with rabbit serum albumin (TZ‐RSA). These tartrazine sensitive patients, however, have lower levels of ‘nonspecific’ IgD than controls, i.e., IgD that reacts with cellulose discs conjugated to phenylsulphonic acid antigenic determinants but does not react with TZ‐RSA. Although it is significant that the observations demonstrate an association of raised specific IgD levels in patients relative to controls, the possible presence in controls of IgD antibodies against other tartrazine antigenic determinants which may play a role in ameliorating or preventing allergic responses should be considered.Anti‐tartrazine IgD or IgE antibody was determined by assay of the IgD or IgE binding to paper discs coupled to a tartrazine antigen analog (AP‐discs). Each result was compared with the amount of antibody binding if the patient's or control serum was preincubated with a free soluble tartrazine‐protein conjugated antigen, before being tested on the discs coupled with antigen (AP‐discs). Reduction of the amount of IgD or IgE binding to the disc‐coupled antigen, when the sera were preincubated with free soluble antigen, indicated specificity of the antibodies for tartrazine antigenic determinants.The specificity of the serum anti‐tartrazine antibodies assayed appears to be the phenylsulphonic moiety, suggesting that the sensitizing agent responsible need not have been tartrazine but could have been one or more of the many other synthetic environmental chemicals with a sulphonated benzene ring. The results obtained suggest that diagnostic tests may be developed for tartrazine hypersensitivity and for hypersensitivity to other chemicals.