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Total Versus Unspecific Binding: Standardization and Optimization of Receptor-Ligand Binding Assays

Intra- and interassay imprecision was evaluated in 3 in vitro ligand binding assays using different types of filtering devices, glass fiber filters and variations in methodology. In the [3H]-spiroperidol test, the already low unspecific binding seemed to be dependent on the type of filters employed, whereas in the [3H]-ketanserin- and [3H]-GR 65630-binding tests, differences were within the range of the normal variabilities. However, in the latter test, which is problematic owing to the high unspecific binding of [3H]-GR 65630, it was found that although the percentage of specific binding was fairly constant on different sheets, large differences in the absolute amounts of total and unspecific binding were observed on consecutive sheets in the same experiment. Thus, it is critically important to filter samples for total and unspecific binding together on the same filter sheet for the calculation of specific binding. Under these precautions, highly reproducible results for IC50-values in the screening of potential 5HT3-receptor ligands were obtained in spite of using rat entorhinal cortex, a relatively large area with suboptimal 5HT3-receptor density. In contrast, when using rat area postrema, which is optimal with respect to receptor density, more than 10 times the number of rats is necessary for a competition experiment due to the small size of this brain part. Since IC50-values for both areas compare favorably, entorhinal cortex should be used for ethical reasons and to minimize costs.