Filter membrane assay

Significant interlaboratory differences in permeability measurements are observed with cell-based assays. It is important to standardize culture conditions and characterize a cell line within one s own laboratory. Permeability differences can be attributed to a number of factors, for example, heterogenecity of cell line, passage number, culture conditions, characteristics of the filter membrane, age of mono-layers and level of differentiation and experimental methodology used. Active... 

Various concentrations of morusin ( ) or TPA (o) were incubated with a particulate fraction of mouse skin in the presence of 4 nmol/L [3H]TPA for 2 h at 4°C, and the assay mixture was filtered on glass filter membrane with acetone cooled in a dry ice-ethanol bath. Non-specific bindings were measured in the presence of 500-fold excess of unlabelled TPA. 

Membrane vesicle suspension should be thawed quickly at 37 °C and stored on ice for about 40 min before use. The transport assay is initiated at 37 °C by addition of membrane suspension (30 pg protein) to the transport assay mixture (110 pi). 20 pi aliquots are removed after 30 or 60 sec intervals, immediately diluted with 1 ml of ice-cold incubation buffer and immediately filtered through nitrocellulose membrane using the vacuum of the filtration apparatus (200 mbar). Filter membranes are rinsed twice with 5 ml of cold incubation buffer, dried and dissolved in 10 ml scintillation fluid to count for radioactivity. 

Abdel-Hamid et al. [122] used a flow-injection amperometric immunofll-tration assay system for the rapid detection of total E. coli and Salmonella. Disposable porous nylon membranes served as a support for the immobilization of anti- ]. coli or anti-Salmonella antibodies. The assay system consists of a flow-injection system, a disposable filter-membrane, and an amperometric sensor. A sandwich immunoassay specifically and directly detected 50 cells ml total E. coli or 50 cells ml Salmonella. The immunosensor can be used as a highly sensitive and automated bioanalytical device for the rapid quantitative detection of bacteria in food and water. 

The Assessment of DNA Crosslinking by Alkaline Elution. DNA damage, that is, interstrand crosslinks, DNA-protein crosslinks, and strand breaks, was determined using the alkaline elution technique (7, ). Cells labeled with C-thymidine for 20-24 hr were deposited on a membrane filter and lysed with a detergent-containing solution. An alkaline solution (pH 12.1-12.2) was then slowly pumped through the filter, and fractions were collected to determine the rate of release of DNA from the filter. For assay of crosslinks, the cells were exposed to x-ray at 0 C prior to deposition on the filter. In order to improve quantitation, control cells labeled with H-thymidine and x-irradiated at 0 were mixed with the experimental Relabeled cells prior to deposition on the filters. The elution of H-DNA serves as an internal reference for normalization of the elution of C-DNA. 

The parallel artificial membrane assay (PAMPA) was first described by Kansy et al. The assay consists in measuring the rate of transfer of compounds from a donor to an acceptor compartment that is separated by a porous filter coated with a mixture of phospholipids in dodecane. The exact nature of the membrane is not known and is unlikely to be a well-defined phospholipid bilayer. Interesting correlations between kinetics of transfer and fraction absorbed in humans were, however, observed. The technology was further improved by taking into account membrane retention and the derivation of differential equations to convert flux ratios into permeability values.The system is now available in the form of a commercial instrument (PSR evolution series, pION, Inc www.pion-inc.com). Improved correlations with GIT permeability were oteerved using different lipid compositions designed to closer mimic the nature of native membranes. ... 

PDA coatings on filter membranes were prepared and used in assays as previously described 9,12). Antibodies were conjugated to PDA coating surfaces with carboxylic acids incorporated using EDC/sulfo-NHS according to the literature (75). Coatings were stored at 4 °C. Attached liposomes were prepared and used in assays as previously described 14). 

Filter paper assays. Phosphorylated membranes were pelleted by centrifugation at 12000 g for 3 min and aliquots of the peptide containing supernatant spotted onto 2 cm x 1 cm Whatman P81 paper strips. After drying at room temperature the strips were washed for 4x5 min in IM phosphoric acid/10% (w/v) trichloroacetic acid and 1x5 min in absolute ethanol. The strips were dried again at room temperature and counted for radioactivity in 2 ml Optiphase Safe (LKB) liquid scintillation cocktail. 

Using a membrane filter binding assay, the competition between poly(ADP-ribose) and DNA for binding of individual histones was measiued (3). The size of the poly(ADP-ribose) molecules firom rat liver nuclei ranged from 10 to 60 monomer units (3). Fig. 1 shows the effect of unlabeled... 

In the presence of CH2-H4folate, FdUMP forms a specific, stable complex with thymidylate synthetase in which all components are covalently bound as depicted in Fig. 1. The affinity constant of this complex is suflSciently high that, with typical concentrations of components used in most experiments, the limiting reagent (FdUMP or enzyme) is completely bound. Using [ H]FdUMP of high specific activity, low levels of complexes present in solution may conveniently he assayed by retention on nitrocellulose filter membranes under conditions in which the free nucleotide is readily removed. The radioactivity remaining on the filter is determined to quantitate the complex. Other conventional methods (e.g., gel filtration, charcoal adsorption of free FdUMP, protein precipitation) may be used for this purpose, but are more tedious and apparently less eflicient. There are expectedly few proteins that will form isolable... 

Methods to Detect and Quantitate Viral Agents in Fluids. In order to assess the effectiveness of membrane filtration the abihty to quantitate the amount of vims present pre- and post-filtration is critical. There are a number of techniques used. The method of choice for filter challenge studies is the plaque assay which utilizes the formation of plaques, localized areas in the cell monolayer where cell death caused by viral infection in the cell has occurred on the cell monolayer. Each plaque represents the presence of a single infectious vims. Vims quantity in a sample can be determined by serial dilution until the number of plaques can be accurately counted. The effectiveness of viral removal may be determined, as in the case of bacterial removal, by comparing the vims concentration in the input suspension to the concentration of vims in the effluent. 

Both multiple-tube and membrane-filter methods are also available for testing for the fecal streptococcal group (20). These assays can be used to provide supplementary data regarding the bacteriological quaUty of water. Other fecal indicators should also be used concurrendy because of the survival characteristics of the fecal streptococci. 

Experiments in 500 ml Erlenmeyer flasks and Fernbach flasks contained 200 ml and 1 L of EPl and EP2 medium respectively. Inocuia added to these cultures was 2 ml of spore suspension (5.0 optical density at 540 nm) for each 100 ml EP medium. All cultures were grown at 37°C in a shaking incubator (New Brunswik Sci. Co., USA), at 200 rpm. Then 10 ml of sample were withdrawn each 24 h during fermentation and immediately filtered through Millipore membranes of 0.45 pm pore size these cell-free filtrates were used for enzymatic assays and extracellular protein determinations by the Lowry method (14). Experiments in the 14 L fermentor (Microgen Fermentor New Brunswik Sci. Co., USA) were carried with lOL of fermentation medium EP2 and inoculum added was IL of mycelium grown 24 h in... 

The equations used to calculate permeability coefficients depend on the design of the in vitro assay to measure the transport of molecules across membrane barriers. It is important to take into account factors such as pH conditions (e.g., pH gradients), buffer capacity, acceptor sink conditions (physical or chemical), any precipitate of the solute in the donor well, presence of cosolvent in the donor compartment, geometry of the compartments, stirring speeds, filter thickness, porosity, pore size, and tortuosity. 

Four neutral lipid models were explored at pH 7.4 (1) 2% wt/vol DOPC in dode-cane, (2) olive oil, (3) octanol, and (4) dodecane. Table 7.5 lists the effective permeabilities Pe, standard deviations (SDs), and membrane retentions of the 32 probe molecules (Table 7.4). The units of Pe and SD are 10 6 cm/s. Retentions are expressed as mole percentages. Figure 7.22a is a plot of log Pe versus log Kd (octanol-water apparent partition coefficients, pH 7.4) for filters loaded with 2% wt/vol DOPC in dodecane (model 1.0, hlled-circle symbols) and with phospholipid-free dodecane (model 4.0, open-circle symbols). The dashed line in the plot was calculated assuming a UWL permeability (see Section 7.7.6) Pu, 16 x 10-6 cm/s (a typical value in an unstirred 96-well microtiter plate assay), and Pe of 0.8 x 10-6 cm/s... 

Faller and Wohnsland [18, 19] developed the PAMPA assay using phospholipid-free hexadecane, supported on 10 pm-thick polycarbonate filters, and were able to demonstrate interesting predictions. Their PAMPA method appeared to be a satisfactory substitute for obtaining alkane/water partition coefficients, which are usually very difficult to measure directly, due to the poor solubility of drug molecules in alkanes. Apparently, membrane retention was not measured. 

The use of artificial membranes to investigate passive permeation processes has a long history, going back more than 40 years [68], The parallel artificial membrane permeation assay (PAMPA) is an application of the filter-supported lipid membrane system [149] and was first introduced by Kansy and... 

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