Membrane-based assays

Stults, N. L., et al. (1992). Use of recombinant biotinylated aequorin in microtiter and membrane-based assays Purification of recombinant aequorin from Escherichia coli. Biochemistry 31 1433-1442. 

Among the membrane-based assay systems, the ATPase assay can be used to identify ABC substrates, since ABC transporters require ATP to transport substrates across the cell membrane. Using isolated membranes containing the ABC transporter of interest or reconstituted ABC protein preparations, ABC substrates would be revealed by an observed increase in ATPase activity (colorimetric detection of inorganic phosphate). In an alternative, inhibition-type (indirect) setup, the test compound is added to a well-established ABC substrate, which creates high ATPase activity. If the test compound is also an ABC substrate, the increased ATPase activity will decrease. 

One major advantage of the glow-type emission obtained from dioxetane enzyme substrates is that one can easily record the emission on photographic film (in much the same way that emission is recorded) to obtain qualitative or semi-quantitative data. This technique has been described for a dry-format membrane-based assay for human luteinizing hormone (hLH), in which Polaroid 612 film (ASA 20,000) was used to record the intensity from unknown samples and from a set of standards (B18). A dioxetane-based ELISA method for human growth hormone (somatotropin) has been recently described. The chemiluminescent assay was reported to be superior to a similar fluorescence assay, and the detection limit for hGH was found to be 5 pM (A9). 

Membrane-Based Assays Membranes prepared from cells expressing transporters have been widely used to study the function of ABC efflux pumps and to identify their substrates or inhibitors. Currently, there are two major membrane-based assays the ATPase assay and the membrane vesicular transport (uptake) assay. Compared to the cell-based assay, the membrane-based assay has several advantages including (1) the assay can be used to characterize the effect of a xenobiotic on one specific efflux transporter (2) the assay can be easily employed in a high throughput mode (3) membranes are easy to be maintained after preparation and (4) the assay is easy to conduct. 

A different renewable solid surface is this employed in membrane-based assays where the membrane allows the covalent immobilization of the reagents, giving rise to a more stable and reproducible assay. The membrane is brought into contact with a SPE so that the immunoassay can be directly transduced [30]. 

Ruell, J. A., Membrane-based drug assays. Modern Drug Disc. Jan., 28-30 (2003). 

This book is written for the practicing pharmaceutical scientist involved in absorption-distribution-metabolism-excretion (ADME) measurements who needs to communicate with medicinal chemists persuasively, so that newly synthesized molecules will be more drug-like. ADME is all about a day in the life of a drug molecule (absorption, distribution, metabolism, and excretion). Specifically, this book attempts to describe the state of the art in measurement of ionization constants (p Ka), oil-water partition coefficients (log PI log D), solubility, and permeability (artificial phospholipid membrane barriers). Permeability is covered in considerable detail, based on a newly developed methodology known as parallel artificial membrane permeability assay (PAMPA). 

There are several approaches to estimating absorption using in vitro methods, notably Caco-2 and MDCK cell-based methods or using methods that assess passive permeability, for example the parallel artificial membrane permeation assay (PAMPA) method. These are reviewed elsewhere in this book. The assays are very useful, and usually have an important role in the screening cascades for drug discovery projects. However, as discussed below, the cell-based assays are not without their drawbacks, and it is often appropriate to use ex vivo and/or in vivo absorption assays. 

Seiler K., Wang K., Bakker E., Morf W.E., Rusterholz B., Spichiger U.E., Simon W., Characterization of sodium-selective optode membranes based on neutral ionophores and assay of sodium in plasma, Clinical Chemistry 1991 37 1350-1355. 

As well as fluorescence-based assays, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In a commercial BIA-core system [231] a hydrophobic SPR sensor with an alkane thiol surface was incubated with vesicles of defined size distribution generating a hybrid membrane by fusion of the lipid vesicles with the alkane thiol layer [232]. If the vesicles contain biotinylated lipopeptides their membrane anchoring can be analyzed by incubation with streptavidine. Accordingly, experiments with lipopeptides representing the C-terminal sequence of N-Ras show clear differences between single and double hydrophobic modified peptides in their ability to persist in the lipid layer [233]. 

The results summarized above were obtained by using fluorescence based assays employing phospholipid vesicles and fluorescent labeled lipopeptides. Recently, surface plasmon resonance (SPR) was developed as new a technique for the study of membrane association of lipidated peptides. Thus, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In SPR (surface plasmon resonance) systemsI713bl changes of the refractive index (RI) in the proximity of the sensor layer are monitored. In a commercial BIAcore system1341 the resonance signal is proportional to the mass of macromolecules bound to the membrane and allows analysis with a time resolution of seconds. Vesicles of defined size distribution were prepared from mixtures of lipids and biotinylated lipopeptides by extruder technique and fused with a alkane thiol surface of a hydrophobic SPR sensor. 

Bagley, D.M., Dong, B.M. and De Salva, S.J. (1989). Assessing the eye irritation potential of surfactant-based materials using the chorioallantoic membrane vascular assay (CAMVA). In Alternative Methods in Toxicology, Vol. 7 (Goldberg, A.M., ed.) Mary Arm Liebert, New York, pp. 265-272. 

Solubility and permeability were measured by a high throughput solubility assay and parallel artificial membrane permeation assay (PAMPA), respectively [56], The assays categorized 14 out of 18 drugs based on the BCS consistent with their known solubility and permeability characteristics [56],... 

One decade has passed since the parallel artificial membrane permeation assay (PAM PA) was first introduced in 1998 [47]. Since then, PAM PA rapidly gained wide popularity in drug discovery [3, 48-51]. Today, PAMPA is the most widely used physicochemical membrane permeation model. The term PAMPA is nowusedas the general name for a plate-based (HTS enabled), biter-supported (filter immobilized) artificial membrane. Typically, phospholipids dissolved in an organic solvent are impregnated into the filter to construct a PAMPA membrane. 

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... 

Common immunochemical assay formats to select from include the 96-well microtiter plates, dipsticks, coated test tubes, and membrane-based flow through devices. If the end-user is a trained technician working in a well-equipped laboratory and needs to detect and tentatively identify, for example, antimicrobial residues in hundreds of meat samples per day, a multiwell or other high-through-put format should be chosen. If, on the other hand, the end user is a quality control inspector at a milk factory who has limited time to find out whether the penicillin residues in the milk waiting to be unloaded exceed a certain level, the same reagents used in the first instance may require a more user-friendly format such as dipstick or membrane-based flow through device. 

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