Methodological considerations for the human platelet 5-HT2A receptor binding kinetic assay

Analysis of an extensive database of human platelet 5-HT2A receptor binding assays has been conducted in order to identify factors that may affect the assay results. Despite anecdotal reports that storage of frozen platelet pellets may affect 5-HT2A binding affinity and capacity, no quantitative study has been reported in the literature. Analysis of binding data for 373 frozen samples with a storage time up to three years is presented in this paper. It is shown that prolonged storage significantly decreases binding. The loss of binding capacity begins in the first six month of storage. Bmax declines by half after 17 month. The impact of storage time on the binding affinity is much smaller. There is only about 20% increase in the value of affinity KD during the half-life of Bmax. Differences in sample storage time may partly explain discrepancies in results between different research groups. Nonspecific binding due to binding to filter material diminishes accuracy and reliability of the binding assays as a result of a decrease in the ratio of specific to nonspecific ratio. A data analysis based on our suggested mathematical model shows that this effect depends on tissue concentration in test tube and becomes pronounced when the concentration is below 0.1 mg protein ml (at 0.2 nM of ligand). Above 0.1 mg protein ml , percentage of specific to total binding exceeds 65%, which is an acceptable level for the ratio. The majority of the binding studies reported in the literature employed a tissue concentration more than 0.5 mg ml , well above the minimal limit sufficient for a reliable assay. However, development of microassays to conserve precious tissue must take the limit into consideration.