Receptor-Based Assays

As previously discussed, there is a tendency (e.g. in Europe) to reduce the amount of certain compounds (e.g. MSG, salt). Current research aims at identifying alternatives for these molecules without compromising quality. By applying state-of-the-art screening tools, such as taste dilution analysis (TDA) [16], receptor-based assays [17] and molecular modelling [18[, a number of MSG alternatives have recently been reported (Fig. 5.51). 

Taste research has evolved considerably over the last years what will certainly result in new compounds and lead structures. The three approaches mentioned, i.e. TDA, receptor-based assays, and molecular modelling, are complementary methods (or tools) that may reveal new taste-active and taste-modifying compounds. The TDA method may especially help discovering taste modulators, because the corresponding receptors and processes are largely unknown. However, many other parameters must be checked, apart from technical taste testing, to evaluate the commercial potential of these new molecules, i.e. stability, safety, cost, availability, range of application, etc. Therefore, just a few molecules may succeed to be widely applied to culinary products. 

Modem methods for the synthesis of drug candidates normally involve combinatorial chemistry techniques and the preparation of libraries of compounds that often have related structures. These libraries are usually dissolved in dimethylsulphoxide (DMSO) solution and archived, from where they are used in high-throughput, in vitro screening procedures for activity against enzyme- or receptor-based assay systems. DMSO is used because it dissolves a wide variety of different compound types, including compounds that are practically insoluble in water it exhibits relatively low toxicity to most types of cells and has low chemical... 

Functional assays that measure parameters reflecting the activity of the toxin can also show some potential for PSP toxin determination. Among these are in vitro tissue culture bioassays, membrane potential-sensitive methods, and receptor-based assays [6], Their major advantage is the possibility of relating the parameter being measured with the sample toxicity. 

Previous studies (Velez et al." ) indicated a good correlation between receptor-based assay (RBA) results and mouse i.p. toxicity (/ =. 97 V=41). In routine work performed between 1995 and 1997, the practical detection limit was 0.04 pg STX eq./lOO g, three orders of magnitude lower than the MBA, providing quantitative data below the detection level of the MBA and thus early warning measurements. Typically, 20-40 samples plus the standard curve, three internal positive controls and 1 negative control could be run per work shift using a conventional liquid scintillation counter. 

All brevetoxins can be detected by immunological techniques, including RIA as well as immunofluores-cent tests and immunoabsorbant assays. The specific binding of brevetoxins and ciguatoxins at a particular site of the voltage-dependent sodium channel can be exploited and these receptors can be used in simple and effective receptor-based assays very much like antibody tests. The receptor assay works in membrane preparations, in microtiter plates, and in solubilized form, and can be done with tissue extracts or biological fluids as toxin source. The system should be also approachable by affinity techniques,... 

A.D. Strosberg, J.E. Leyseng, Receptor-based assays, Curr. Opin. Biotechnol, 2, 30 (1991). 

The enhanced availability of receptors, enzymes and cell systems has permitted these entities to be considered as viable means for the evaluation of very small amounts of a sample on an automated basis. This, in turn, has led to another important change, namely the rate at which new biologically active entities can be discovered. Whereas it previously took weeks or months to test a few hundred samples, it now takes, for some assays, only a matter of hours. Whole cell, enzyme-based, and receptor-based assays are now quite routine in many therapeutic target areas, and the emphasis is shifting rapidly to genetically-engineered assays which evaluate the ability of a compound to interfere with a biological process in an exceptionally specific manner. 

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