Filter-based assays

Variations on the filter-based assay have been designed to approximate more physiological contexts. Such assays include tumor cell invasion across a confluent cell monolayer (e.g., endothelial cells (EC) as a surrogate for intravasation or extravasation during hematogenous metastasis (24)) and ovarian carcinoma invasion of mesothelial cell monolayers (25). Additionally, 1 mm thick slices of human brain tissue have been used as a tissue barrier on Transwell filters with invasion of GFP-labeled glioma cells measured by confocal microscopy (26). 

Kinetic solubility This pragmatic approach starts with a concentrated compound solution in pure DM SO further diluted in a buffer medium. The amount of compound in solution is measured after a few minutes incubation either by recording its UV absorbance (with or without a chromatographic step) or precipitate formation using an optical method (turbidimetry, nephelometry or flow cytometry). This approach mimics the typical path of the compound in biochemical, cellular assays or in vivo animal models. Kinetic solubility usually serves as a quality filter prior to cell based assays (see paragraphs on solubility, permeability and cellular assays). 

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... 

The modified cellulose filter binding assay is based on the tight binding of proteins to this kind of filter material. When a protein-nucleic acid mixture is filtered, proteins are retained on the filter while nucleic acids are washed through. However, nucleic acids are also retained on the filter if they are bound to proteins. Thus, free and protein-bound nucleic acids can be separated [43]. 

Total cellulolytic activity was measured using the filter paper assay (FP A) according to Ghose (11) based on an estimation of the released reducing sugars by dinitrosalicylic (DNS) acid (12). 

Monochromator-based detection, however, can perform spectral scanning that is useful for the assay development and for spectral determinations of fluorophores and compounds. Monochromator types eliminate the need to buy different filter sets for each wavelength. Our laboratory uses several filter-based plate readers and one monochromatic type to accommodate the needs of assay development and compound screening (Table 11.2). 

A broad variety of particle sizes are available, from microspheres with less than 5-pm particle size up to resins of 700-pm particle size. The large beads can be used as polymeric microreactors.P l As the separation between resin and liquid is still performed by filtration, different glass filters have to be used the porosity and nominal pore size of these filters are listed in Table 5. In those applications where single beads are used (split/mix, single-bead analysis, bead-based assays) it is essential to know the capacity of the single beads. For practical purposes a correlation between bead size and capacity per bead is reported in Table 6. 

Another example concerns the immobilization of a biocatalyst—the enzyme glucose oxidase (GOD)—to the silica nanotubes [4]. GOD was immobilized, on both the inside and outside surfaces, via the aldehyde silane route. These GOD-nanotubes (60 nm diameter) were dispersed into a solution containing 90 mM glucose and also the components of the standard dianisidine-based assay for GOD activity. A GOD activity of 0.5 0.2 units per milligram of nanotubes was obtained. These studies also showed that protein immobilized via the Schiff base route is not leached from the nanotubes, where GOD activity ceased when the nanotubes were filtered from the solution. 

The most rugged instruments available, filter-based devices are capable of performing rather sophisticated analyses. With a filter wheel providing several dozen wavelengths of observation, multiple-analyte analyses are quite common. These multiple assays are more amenable to agricultural or food products than pharmaceuticals, if only for validation purposes. Fine chemicals, pharmaceuticals, and gasses may be likely to have sharper peaks and are better analyzed via a continuous monochromator (i.e., grating, FT, AOTF). 

In contrast to cell-based assays, noncell-based permeability assays using artificial membranes supported on filters are fast, flexible, cheap, and fully automatable. They are therefore ideally placed for use in high throughput. There have been many variations of this assay in terms of the fine details of the experiment and these will be discussed in due course however, the basic principles remain the same, based on a 96-well microtiter plate format. 

In another approach, a filter-binding assay was found to be of value to study the kinetics of triplex formation by 19-mers in the R RY motif [40]. It was found that the Kojf is pH dependent owing to a rapid acid-base equilibrium of... 

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